Systems, methods, and compositions for correction of frameshift mutations

ABSTRACT

The disclosure provides systems, methods, and compositions for a target specific nuclease and a blunting enzyme to correct frameshift mutations for genome editing and treatment of diseases. In some embodiments, the target specific nuclease and the blunting enzyme are combined with a guide RNA and/or a microhomology-mediated end joining (MMEJ) inhibitor.

RELATED APPLICATIONS

This application is a continuation of U.S. Application No. 17/067,379, filed on Oct. 9, 2020, which claims the benefit of U.S. Provisional Application No. 62/913,048 filed on Oct. 9, 2019 and U.S. Provisional Application No. 62/984,422, filed on Mar. 3, 2020, the entire disclosures of which are hereby incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was made with government support under Grant No. U01CA250554 awarded by the U.S. National Institute of Health (NIT)/National Cancer Institute (NCI) Next Generation of Cancer Model (NGCM) program. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on Jan. 24, 2023, is named 735898_083474-011USCON.xml and is 307,000 bytes in size.

FIELD

The subject matter disclosed herein is generally related to systems, methods, and compositions for correction frameshift mutations, accurate genome editing and treatment of diseases.

BACKGROUND

Frameshift mutations are genetic mutations that are caused by insertion or deletion (indels) of nucleotides in a coding region of a nucleic acid sequence that is not divisible by three. The indel results in mutated sequences that, due to the triplet nature of gene expression by codons, changes the reading frame of the codon and therefore change the translation of the nucleic acid sequence.

Frameshift mutations are present in number of diseases, but genetic treatments for these diseases are limited. They often involve removing large section from a genome sequence and lead to undesired side effects.

Therefore, there is need for more efficient tools to correct frameshift mutations.

SUMMARY

The present disclosure provides systems, methods, and compositions for correction frameshift mutations, accurate genome editing and treatment of diseases.

The present disclosure provides a composition, which comprises a target specific nuclease, wherein the target comprises a double stranded DNA (dsDNA), and a double strand break (DSB)-end blunting enzyme. The target specificity of the nuclease can be provided by a guide RNA (gRNA). The gRNA can be a single guide RNA (sgRNA). The sgRNA can comprise a nucleic acid sequence at least 75% identical to the nucleic acid sequence of SEQ ID NOs: 54-64. If desired, the composition can further comprise a MS2-binding protein, wherein the sgRNA can comprise one or more MS2 stem loops, and wherein the MS2-binding protein can be linked to the sgRNA by the one or more MS2 stem loops and can bind to the DSB-end blunting enzyme. If desired, the nuclease predominantly can induce staggered ends on the cleaved dsDNA. If desired, the nuclease can be an altered scissile variant. If desired, the altered scissile variant can be ΔF916, LZ3Cas9 (N690C, T769I, G915M, N980K), G915F, F916P, R918A, R919P or Q920P. If desired, the nuclease can be selected from the group consisting of SpCas9, LbCasl2a, AsCas12a and FnCasl2a.

In some embodiments, the nuclease can comprise an amino acid sequence at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the nuclease can comprise an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the nuclease can comprise an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the nuclease can comprise an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the nuclease can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the amino acid sequence can specifically bind to a protospacer-adjacent motif (PAM). The PAM can be selected from the group consisting ofNNNNGATT, NNNNGNNN, NNG, NG, NGAN, NGNG, NGAG, NGCG, NAAG, NGN, NRN, NNGRRN, NNNRRT, TTTN, TTTV, TYCV, TATV, TYCV, TATV, TTN, KYTV, TYCV, TATV, and TBN.

In some embodiments, the DSB-end blunting enzyme can be a polymerase. The polymerase can be selected from the group consisting of DNA polymerase λ (POLL), DNA polymerase µ (POLM), DNA polymerase β (POLB), DNA polymerase γ (POLG), DNA polymerase τ (POLI), DNA polymerase η (POLH), TENT4A, DNA polymerase v (POLN), DNA Ligase 4, DNTT, XRCC4, DNA Polymerase IV, fungi pol IV-like DNA polymerase, DNA polymerase/3′-5′ exonuclease Pol X, and T4 DNA polymerase (T4pol).

In some embodiments, the DSB-end blunting enzyme can be a single-strand DNA specific nuclease. The single-strand DNA specific nuclease can be selected from the group consisting of MGME1, FEN1, DNA2, XRN2, EXOG, EXOS, AP endonuclease, RecJ exonuclease (RecJ), XseA, XseB, S1 nuclease (nucS), P1 nuclease, Artemis, T4 DNA polymerase (T4pol), and Csml.

In some embodiments, the DSB-end blunting enzyme can be covalently bound to the nuclease by a linker. The linker can be a peptide.

In some embodiments, the dsDNA can be in a cell. The cell can be a eukaryotic cell. The eukaryotic cell can be a mammalian cell. The mammalian cell can be a human cell.

In some embodiments, the composition can further comprise an inhibitor of the microhomology-mediated end joining (MMEJ) pathway. The MMEJ pathway inhibitor can be a CtIP or MRN inhibitor. The CtIP inhibitor can be selected from KLHL15 and PIN1. The MRN inhibitor can be selected from Elb55K and E40rf6.

In some embodiments, a first nucleic acid molecule encoding the nuclease is disclosed.

In some embodiments, a second nucleic acid molecule encoding the DSB-end blunting enzyme is disclosed.

In some embodiments, a third nucleic acid molecule encoding the sgRNA is disclosed.

In some embodiments, one or more vectors comprising the nucleic acid molecule are disclosed.

In some embodiments, a cell comprising the composition, the nucleic acid molecule or the one or more vectors is disclosed. If desirable, the cell can be a prokaryotic cell. If desirable, the cell can be a eukaryotic cell. If desired, the eukaryotic cell can be a mammalian cell. If desired, the mammalian cell can be a human cell.

In some embodiments, a method of inserting or deleting one or more single base pairs in a double-stranded DNA (dsDNA) is disclosed, the method comprises cleaving the dsDNA at a target site with a target specific nuclease, wherein the cleavage results in overhangs on both dsDNA ends, inserting a nucleotide complementary to the overhanging nucleotide on both of the dsDNA ends using a double strand break (DSB)-end blunting enzyme, or removing the overhanging nucleotide on both of the dsDNA ends using the DSB-end blunting enzyme, and ligating the dsDNA ends together, thereby inserting or deleting a single base pair in the dsDNA. The target specificity of the nuclease can be provided by a guide RNA (gRNA). The gRNA can be a single guide RNA (sgRNA). The sgRNA can comprise a nucleic acid sequence at least 75% identical to the nucleic acid sequence of SEQ ID NOs: 54-64. The sgRNA can comprise one or more MS2 stem loops that link a MS2-binding protein to the sgRNA, and wherein the MS2-binding protein can bind to the DSB-blunting enzyme. The DSB-end blunting enzyme can be overexpressed. The nuclease can induce staggered ends on the cleaved dsDNA. The nuclease can be an altered scissile variant. The altered scissile variant can be ΔF916, G915F, F916P, R918A, R919P or Q920P. The nuclease can be selected from the group consisting of SpCas9, LZ3Cas9 (N690C, T769I, G915M, N980K), LbCasl2a, AsCasl2a and FnCasl2a.

In some embodiments, the nuclease of the method can comprise an amino acid sequence at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the nuclease of the method can comprise an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the nuclease of the method can comprise an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the nuclease of the method can comprise an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the nuclease of the method can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the amino acid sequence of the method can specifically bind to a protospacer-adjacent motif (PAM). The PAM can be selected from the group consisting of NNNNGATT, NNNNGNNN, NNG, NG, NGAN, NGNG, NGAG, NGCG, NAAG, NGN, NRN, NNGRRN, NNNRRT, TTTN, TTTV, TYCV, TATV, TYCV, TATV, TTN, KYTV, TYCV, TATV, and TBN.

In some embodiments, the DSB-end blunting enzyme of the method can be a polymerase. The polymerase can be selected from the group consisting of DNA polymerase λ (POLL), DNA polymerase µ (POLM), DNA polymerase (3, DNA polymerase γ (POLG), DNA polymerase τ (POLI), DNA polymerase η (POLH), TENT4A, DNA polymerase v (POLN), DNA Ligase 4, DNTT, XRCC4, DNA Polymerase IV, fungi pol IV-like DNA polymerase, DNA polymerase/3′-5′ exonuclease Pol X, and T4 DNA polymerase (T4pol).

In some embodiments, the DSB-end blunting enzyme of the method can be a single-strand DNA specific nuclease. The single-strand DNA specific nuclease can be selected from the group consisting of MGME1, FEN1, DNA2, XRN2, EXOG, EXOS, AP endonuclease, RecJ exonuclease, XseA, XseB, S1 nuclease (nucS), P1 nuclease, Artemis, T4 DNA polymerase (T4pol), and Csml. The DSB-end blunting enzyme can be covalently bound to the nuclease by a linker. The linker can be a peptide.

In some embodiments, the dsDNA of the method can be a cell. The cell can be a eukaryotic cell. The eukaryotic cell can be a mammalian cell. The mammalian cell can be a human cell.

In some embodiments, the method can further comprise an inhibitor of the microhomology-mediated end joining (MMEJ) pathway. The MMEJ pathway inhibitor can be a CtIP or MRN inhibitor. The CtIP inhibitor can be selected from KLHL15 and PIN1. The MRN inhibitor can be selected from Elb55K and E40rf6.

In some embodiments, a method of treating a disease caused by a frameshift mutation in the dsDNA in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition, the nucleic acid molecule, the vector or the cell is disclosed.

In some embodiments, a method of treating a disease caused by a frameshift mutation in the dsDNA in a subject in need thereof comprising inserting or deleting a single base pair in the dsDNA with the frameshift mutation according is disclosed.

In some embodiments, a method of enhancing out-frame mutation in the dsDNA in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition, the nucleic acid molecule, the vector, or the cell is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects, features, benefits, and advantages of the embodiments described herein will be apparent with regard to the following description, appended claims, and accompanying drawings where:

FIG. 1 is a schematic illustration of a variety of different frameshift mutations according to embodiments of the present teachings;

FIG. 2 is a schematic illustration of how frameshift mutations can be corrected according to embodiments of the present teachings;

FIG. 3A is a schematic representation of a CRISPR-Cas9 and a blunting enzyme connected to the CRISPR-Cas9 by a linker, without the use of a donor template according to embodiments of the present teachings;

FIG. 3B is a schematic representation of a CRISPR-Cas9, a blunting enzyme and a microhomology-mediated end joining (MMEJ) inhibitor without the use of a donor template according to embodiments of the present teachings;

FIG. 3C is a schematic representation of a CRISPR-Cas9, a MS2-loop, MS2-binding protein, and a blunting enzyme according to embodiments of the present teachings;

FIG. 3D is a schematic representation of a CRISPR-Cas9 and a blunting enzyme, without the use of a donor template according to embodiments of the present teachings;

FIG. 4 is a schematic illustration of the process of using SpCas9 to generate both blunted and staggered DNA ends according to embodiments of the present teachings;

FIG. 5 is a schematic illustration of a Cas9 gene editing system resulting in an induction of a precise and predictable mutations without the use of a donor template according to embodiments of the present teachings. The Cas9 gene editing system comprises: a target sequence of the sequences of SEQ ID NO: 118 (acattgtagccctctgtgtgctcaagggggg) and SEQ ID NO: 119 (ccccccttgagcacacagagggctacaatgt); staggered end sequences of the sequences of SEQ ID NO: 120 (acattgtagccctctgtgtgct) and SEQ ID NO: 121 (gagcacacagagggctacaatgt), and SEQ ID NO: 122 (caagggggg) and SEQ ID NO: 123 (cccccctt); blunted end sequences of the sequences of SEQ ID NO: 124 (acattgtagccctctgtgtgct) and SEQ ID NO: 125 (agcacacagagggctacaatgt), and SEQ ID NO: 126 (caagggggg) and SEQ ID NO: 127 (ccccccttg); produce “blunt ends” fill-in sequences of the sequences of SEQ ID NO: 128 (acattgtagccctctgtgtgctc) and SEQ ID NO: 129 (gagcacacagagggctacaatgt), and SEQ ID NO: 130 (caagggggg) and SEQ ID NO: 131 (ccccccttg); produce “blunt ends” chew-back sequences of the sequences of SEQ ID NO: 132 (acattgtagc cctctgtgtgct) and SEQ ID NO: 133 (agcacacagagggctacaatgt), and SEQ ID NO: 134 (aagggggg) and SEQ ID NO: 135 (cccccctt); a repair insertion sequence of the sequence of SEQ ID NO: 136 (acattgtagccctctgtgtgctccaagggggg); and a deletion sequence of the sequence of SEQ ID NO: 137 (acattgtagccctctgtgtgctaagggggg);

FIG. 6 is a schematic illustration of a variety of different frameshift mutations and how the composition in the instant disclosure corrects them according to embodiments of the present teachings;

FIG. 7 is a schematic representation of two double-strand break repair pathways according to embodiments of the present teachings;

FIG. 8 is a schematic representation of the primary structures of family X polymerases according to embodiments of the present teachings;

FIG. 9 is a schematic illustration of a frameshift mutation which is present in Parkinson’s disease according to embodiments of the present teachings. The frameshift mutation comprises a WT sequence of the sequence of SEQ ID NO: 138 (aatccttatgcagaatcagagctcaaa) and a Parkinson disease sequence of the sequence of SEQ ID NO: 139 (aatccttatgcagacagaatcagagct);

FIG. 10 is a schematic illustration of the use of the Cas9 gene editing system to correct a 1 bp BRCA2 frameshift mutation (c.8015_8016insA) and a 2 bp NF1 frameshift mutation (c.2027delC) according to embodiments of the present teachings. The τ bp frameshift editing system comprises a WT sequence of the sequence of SEQ ID NO: 140 (agatcggctataaaaaagataatggaa), a HCT116 (mutant) sequence of the sequence of SEQ ID NO: 141 (agatcggctataaaaaaagataatgga), and a chew-back sequence of the sequence of SEQ ID NO: 142 (agatcggctataaaaaaagtaatggaa). The 2bp frameshift editing system comprises a WT sequence of the sequence of SEQ ID NO: 143 (ggaacccccccgatttgccgacaagcc), a HCT116 (mutant) sequence of the sequence of SEQ ID NO: 144 (ggaaccccccgatttgccgacaagccc), and a fill-in sequence of the sequence of SEQ ID NO: 145 (ggaacccccccgatttgccgacaagcc);

FIG. 11A is a diagram showing the probability distribution of indel mutations in PCSK9 exon 12 when induced by Cas9 only according to embodiments of the present teachings;

FIG. 11B is a diagram showing the probability distribution of indel mutations in PCSK9 exon 12 when induced by Cas9 and DNA polymerase µ (POLM) according to embodiments of the present teachings;

FIG. 11C is a diagram showing the probability distribution of indel mutations in PCSK9 exon 12 when induced by Cas9 and EXOG according to embodiments of the present teachings;

FIG. 1 ID is a diagram showing the probability distribution of indel mutations in PCSK9 exon 12 when induced by Cas9 and T4 DNA polymerase (T4pol) according to embodiments of the present teachings;

FIG. 11E is a diagram showing the probability distribution of indel mutations in PCSK9 exon 12 when induced by Cas9 and DNA polymerase λ (POLL) according to embodiments of the present teachings;

FIG. 11F is a diagram showing the probability distribution of indel mutations in PCSK9 exon 12 when induced by Cas9 and MGME1 according to embodiments of the present teachings;

FIG. 11G is a diagram showing the probability distribution of indel mutations in PCSK9 exon 12 when induced by Cas9 and RecJ exonuclease (RecJ) according to embodiments of the present teachings;

FIG. 11H is a diagram showing the probability distribution of indel mutations in PCSK9 exon 12 when induced by Cas9 and S 1 Nuclease (nucS) according to embodiments of the present teachings;

FIG. 12A is a diagram showing probability distribution of +1 T insertion and indel mutations in PCSK9 exon 12 when induced by Cas9 and POLL, and Cas9 and POLM for a target sequence of the sequences of SEQ ID NO: 146 (ccgactttgtccctctctcagccc) and SEQ ID NO: 147 (gggctgagagagggacaaagtcgg) according to embodiments of the present teachings;

FIG. 12B is a diagram showing probability distribution of -1 deletion, +1 T insertion and indel mutations in PCSK9 exon 12 when induced by Cas9 and T4 polymerase for a target sequence of the sequences of SEQ ID NO: 148 (ccgactttgtccctctctcagccc) and SEQ ID NO: 149 (gggctgagagagggacaaagtcgg) according to embodiments of the present teachings;

FIG. 13A is a diagram showing probability distribution of indel mutations in GYPB gene when induced by Cas9 only according to embodiments of the present teachings;

FIG. 13B is a diagram showing the probability distribution of indel mutations in GYPB when induced by Cas9 and POLM according to embodiments of the present teachings;

FIG. 13C is a diagram showing the probability distribution of indel mutations in GYPB when induced by Cas9 and EXOG according to embodiments of the present teachings;

FIG. 13D is a diagram showing the probability distribution of indel mutations in GYPB when induced by Cas9 and T4pol according to embodiments of the present teachings;

FIG. 13E is a diagram showing the probability distribution of indel mutations in GYPB when induced by Cas9 and POLL according to embodiments of the present teachings;

FIG. 13F is a diagram showing the probability distribution of indel mutations in GYPB when induced by Cas9 and MGME according to embodiments of the present teachings;

FIG. 13G is a diagram showing the probability distribution of indel mutations in GYPB when induced by Cas9 and RecJ according to embodiments of the present teachings;

FIG. 13H is a diagram showing the probability distribution of indel mutations in GYPB when induced by Cas9 and nucS according to embodiments of the present teachings;

FIG. 14A is a diagram showing the probability distribution of indel mutations in TPH2 exon 9 when induced by Cas9 alone according to embodiments of the present teachings;

FIG. 14B is a diagram showing the probability distribution of indel mutations in TPH2 exon 9 when induced by Cas9 and POLM according to embodiments of the present teachings;

FIG. 14C is a diagram showing the probability distribution of indel mutations in TPH2 exon 9 when induced by Cas9 and EXOG according to embodiments of the present teachings;

FIG. 14D is a diagram showing the probability distribution of indel mutations in TPH2 exon 9 when induced by Cas9 and T4pol according to embodiments of the present teachings;

FIG. 14E is a diagram showing the probability distribution of indel mutations in TPH2 exon 9 when induced by Cas9 and POLL according to embodiments of the present teachings;

FIG. 14F is a diagram showing the probability distribution of indel mutations in TPH2 exon 9 when induced by Cas9 and MGME according to embodiments of the present teachings;

FIG. 14G is a diagram showing the probability distribution of indel mutations in TPH2 exon 9 when induced by Cas9 and RecJ according to embodiments of the present teachings; and

FIG. 14H is a diagram showing the probability distribution of indel mutations in TPH2 exon 9 when induced by Cas9 and nucS according to embodiments of the present teachings.

DETAILED DESCRIPTION

It will be appreciated that for clarity, the following disclosure will describe various aspects of embodiments. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

As used herein, the singular forms “a”, “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise.

The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, +/-5% or less, +/-1% or less, +/-0.5% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

The term “staggered end” when it refers to a double stranded DNA (dsDNA) molecule refers to the 5′ and or 3′ ends of that molecule having at least one nucleotide that is not hybridized to the opposite strand of the dsDNA.

The term “blunt end” when it refers to a dsDNA molecule refers to the 5′ and or 3′ ends of that molecule having nucleotides that hybridize to the opposite strand of the dsDNA.

The term “variant” as used herein means a polypeptide or nucleotide sequence that differs from a given polypeptide or nucleotide sequence in amino acid or nucleic acid sequence by the addition (e.g., insertion), deletion, or conservative substitution of amino acids or nucleotides, but that retains the biological activity of the given polypeptide (e.g., a variant nucleic acid could still encode the same or a similar amino acid sequence). A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity and degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (see, e.g., Kyte et al., J. Mol. Biol., 157: 105-132 (1982)). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. The present disclosure provides amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids also can be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (see, e.g., U.S. Pat. No. 4,554,101). Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. The present disclosure provides substitutions are performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. “Variant” also can be used to describe a polypeptide or fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains its biological activity or antigen reactivity. Use of “variant” herein is intended to encompass fragments of a variant unless otherwise contradicted by context. The term “protospacer-adjacent motif” as used herein refers to a DNA sequence immediately following a DNA sequence targeted by a nuclease. Examples of protospacer-adj acent motif include, without limitation, NNNNGATT, NNNNGNNN, NNG, NG, NGAN, NGNG, NGAG, NGCG, NAAG, NGN, NRN, NNGRRN, NNNRRT, TTTN, TTTV, TYCV, TATV, TYCV, TATV, TTN, KYTV, TYCV, TATV, and TBN.

The term “MS2 stem loop” as used herein refers to a pattern in a single stranded nucleotide strand originated from a bacterial virus when two regions of the same strand base-pair to form a double helix that ends in an unpaired loop.

Alternatively or additionally, a “variant” is to be understood as a polynucleotide or protein which differs in comparison to the polynucleotide or protein from which it is derived by one or more changes in its length or sequence. The polypeptide or polynucleotide from which a protein or nucleic acid variant is derived is also known as the parent polypeptide or polynucleotide. The term “variant” comprises “fragments” or “derivatives” of the parent molecule. Typically, “fragments” are smaller in length or size than the parent molecule, whilst “derivatives” exhibit one or more differences in their sequence in comparison to the parent molecule. Also encompassed modified molecules such as but not limited to post-translationally modified proteins (e.g. glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids such as methylated DNA. Also, mixtures of different molecules such as but not limited to RNA-DNA hybrids, are encompassed by the term “variant”. Typically, a variant is constructed artificially, preferably by gene-technological means whilst the parent polypeptide or polynucleotide is a wild-type protein or polynucleotide. However, also naturally occurring variants are to be understood to be encompassed by the term “variant” as used herein. Further, the variants usable in the present disclosure may also be derived from homologs, orthologs, or paralogs of the parent molecule or from artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent molecule, i.e. is functionally active.

Alternatively, or additionally, a “variant” as used herein, can be characterized by a certain degree of sequence identity to the parent polypeptide or parent polynucleotide from which it is derived. More precisely, a protein variant in the context of the present disclosure exhibits at least 80% sequence identity to its parent polypeptide. A polynucleotide variant in the context of the present disclosure exhibits at least 70% sequence identity to its parent polynucleotide. The term “at least 70% sequence identity” is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to a sequence identity of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference polypeptide or to the respective reference polynucleotide.

The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. on www.ebi.ac.uk/Tools/clustalw/ or on www.ebi.ac.uk/Tools/clustalw2/index.html or on npsa-pbil.ibcp.fr/cgi-bin/npsa automat.pl?page=/NPSA/npsa clustalw.html. Preferred parameters used are the default parameters as they are set on www.ebi.ac.uk/Tools/clustalw/ or www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLAS TN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

Overview

Some embodiments disclosed herein provide non-naturally occurring or engineered systems, methods, and compositions for target specific nucleases combined with blunting enzymes to correct frameshift mutations for genome editing and treatment of diseases. Frameshift mutations are genetic mutations that are caused by insertion and deletion (indels) of nucleotides in a DNA nucleic acid sequence that is not divisible by three. Due to the triplet nature of gene expression by codons, the indel can change the reading frame of the codon and therefore change the translation of the gene. Different types of frameshift mutations and examples of in-frame corrections of them are shown in FIGS. 1 and 2 .

In some embodiments, the systems disclosed herein comprise a target specific nuclease, wherein the target comprises a double-stranded DNA (dsDNA) as well as a blunting enzyme. The systems disclosed herein can also comprise targeting moiety and/or a microhomology-mediated end joining (MMEJ) inhibitor.

In some embodiments, the target specific nuclease can be a CRISPR associated protein (Cas). In some embodiments, the targeted nuclease is a Cas9 protein as illustrated in FIGS. 3A-3D. In some embodiments, the blunting enzyme is joined to the targeted nuclease by a linker. In some embodiments, the blunting enzyme is separate from the targeted nuclease. In some embodiments, the composition further comprises a MMEJ inhibitor. In some embodiments, the composition further comprises a single guide RNA (sgRNA). In some embodiments, the composition further comprises a sgRNA and a MS2-binding protein, wherein the sgRNA comprises one or more MS2 stem loops. The MS2-binding protein is linked to the sgRNA by the one or more MS2 stem loops and binds to the blunting enzyme to form a blunting enzyme fused-MS2 binding protein.

The target specific nuclease combined with a blunting enzyme can correct frameshift mutations in genes in cells and tissues. In some embodiments, cells include eukaryotic cells, mammalian cells, and human cells. The target specific nuclease combined with a blunting enzyme can induce one or more single-base insertions and deletions (indels). In some embodiments, the targeted nuclease creates staggered ends when it cleaves the target dsDNA. When the staggered ends are created by the target specific nuclease, a blunting enzyme can be used to ether “fill in” the staggered end with a polymerase or “chew back” the staggered end with a nuclease. Filling in followed by ligation creates a one or more bp insertion and chewing back followed by ligation creates one or more bp deletion. (See FIGS. 4-5 ). In some embodiments, the target specific nuclease and a blunting enzyme induce a precise and predictable mutation in a dsDNA without the use of a donor template.

Microhomology-mediated end joining (MMEJ) is one of the pathways for repairing double-strand breaks in DNA. In MMEJ, microhomologous sequences are used to align broken ends often resulting in deletions flanking the original break. In some embodiments, if a target specific nuclease were used to cleave dsDNA, MMEJ could create an unintended deletion.

Non-homologous end joining (NHEJ) is another pathway for repairing double-strand breaks in DNA. In NHEJ, the broken ends are directly ligated together without use of a homologous template. In some embodiments, if a target specific nuclease were used to cleave dsDNA, NHEJ would directly ligate the cleaved dsDNA without deletions and therefore accurately edit the target sequence. (See FIGS. 6 and 7 ).

In some embodiments, an inhibitor of MMEJ is used to keep cleaved DNA from undergoing MMEJ and being subject to unintended deletion of the sequence of the dsDNA flanking the cleavage.

Target Specific Nucleases

In some embodiments, a target specific nuclease is a nuclease that cleaves a dsDNA and, at least in some cases, leaves a staggered end at the cleavage site. The target specific nuclease disclosed herein can be for example, without limitation, Cas12a, LbCasl2a, FnCasl2a, AsCas12a, Cas9, SpCas9, SaCas9, LZ3Cas9, Casφ, and the double combinations of Cas9 nickase, zinc finger nuclease (ZFN), and TAL Effector Nuclease (TALEN). The LZ3Cas9 disclosed here can be N690C, T769I, G915M, or N980K. In some embodiments, the target specific nuclease cleaves dsDNA in the genome of a cell providing staggered ends. In some embodiments, the target specific nuclease provides a dsDNA cleavage resulting in staggered ends more than 10% of the time. In some embodiments, the target specific nuclease provides a dsDNA cleavage resulting in staggered ends more than 20% of the time. In other embodiments, the target specific nuclease provides a dsDNA cleavage resulting in staggered ends more than 3, 40, 50, 60, 70, 80, 90, 95, or 99% of the time.

In some embodiments, the target specific nuclease is a CRISPR associated protein (Cas). In these embodiments, the Cas uses a guide RNA (gRNA) to provide specificity. In some embodiments, the gRNA is a single guide RNA (sgRNA) i.e., a fusion of two noncoding RNAs: a synthetic CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA).

In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a Cas protein to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies, ELY-KND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.

In some embodiments, the sgRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 54-64. For example, the sgRNA can comprise a nucleic acid sequence at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 54-64.

In some embodiments, the target specific nuclease is Cas9. In some embodiments, the target nuclease is a scissile variant. In some embodiments, the Cas9 is a scissile variant of Cas9. In some embodiments, the scissile is for example, without limitation, ΔF916, LZ3Cas9, G915F, F916P, R918A, R919P, Q920P, N690C, T769I, G915M and N980K. In some embodiments, the LZ3Cas9 is N690C, T769I, G915M, or N980K.

The target specific nuclease can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106. For example, the target specific nuclease comprises an amino acid sequence at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.

In some embodiments, the target specific nuclease is a zinc finger nuclease (ZFN). A single zinc finger contains approximately 30 amino acids and the domain functions by binding 3 consecutive base pairs of DNA via interactions of a single amino acid side chain per base pair. The modular structure of the zinc finger motif permits the conjunction of several domains in series, allowing for the recognition and targeting of extended sequences in multiples of 3 nucleotides. These targeted DNA-binding domains can be combined with a nuclease domain, such as FokI, to generate a site-specific nuclease, called a “zinc finger nuclease” (ZFNs) that can be used to introduce site-specific double strand breaks at targeted genomic loci. This DNA cleavage stimulates the natural DNA-repair machinery, leading to one of two possible repair pathways, NHEJ and HDR. For example, the ZFN can target the Rosa26 locus (Perez-Pinera et al. Nucleic Acids Research (2012) 40:3741-3752) or a dystrophin gene.

In some embodiments, the target specific nuclease is a TAL effector nuclease (TALEN). The TALEN can be used to introduce site-specific double strand breaks at targeted genomic loci. Site-specific double-strand breaks are created when two independent TALENs bind to nearby DNA sequences, thereby permitting dimerization of FokI and cleavage of the target DNA. TALENs have advanced genome editing due to their high rate of successful and efficient genetic modification. This DNA cleavage can stimulate the natural DNA-repair machinery, leading to one of two possible repair pathways: homology-directed repair (HDR) or the non-homologous end joining (NHEJ) pathway. The TALENs can be designed to target any gene involved in a genetic disease.

The TALENs can include a nuclease and a TALE DNA-binding domain that binds to the target gene in a TALEN target region. The target gene can have a mutation such as a frameshift mutation or a nonsense mutation. If the target gene has a mutation that causes a premature stop codon, the TALEN can be designed to recognize and bind a nucleotide sequence upstream or downstream from the premature stop codon. A “TALEN target region” includes the binding regions for two TALENs and the spacer region, which occurs between the binding regions. The two TALENs bind to different binding regions within the TALEN target region, after which the TALEN target region is cleaved. Examples of TALENs are described in International Patent Application No. PCT/US2013/038536, which is incorporated by reference in its entirety.

In some embodiments, the target specific nucleases include tags including for example, without limitation, 3xFlag, nuclear localization sequence (NLS), and the combination of 3xFlag and NLS.

Blunting Enzymes

In some embodiments, the blunting enzyme or double strand break-end blunting enzyme (both terms are used interchangeably herein), is an enzyme that is able either to remove or add nucleotides to a staggered end of a double stranded DNA molecule to produce a blunt end. In some embodiments, the blunting enzyme disclosed herein is a polymerase or a nuclease. In some embodiments, the DSB-blunting enzyme is a single-strand DNA specific nuclease.

In some embodiments, the blunting enzyme is a polymerase selected from polymerase λ (POLL), polymerase µ (POLM), polymerase v (POLN), polymerase η (POLH), polymerase β (POLB), DNA polymerase θ (POLQ), DNA polymerase κ (POLK), DNA polymerase IV (Saccharomyces cerevisiae), DNA polymerase y (POLG), DNA polymerase τ (POLI), DNA polymerase ξ, DNA polymerase v (POLN), DNA nucleotidylexotransferase (DNTT), TENT4A, DNA ligase 4, fungi pol IV-like DNA polymerase (Neurospora crassa), DNA polymerase/3′-5′ exonuclease PolX (Bacillus subtilis), Family X DNA Polymerase (Deinococcus radiodurans), and T4 DNA polymerase (Scherichia virus T4). In some embodiments, the blunting enzyme is a nuclease. In some embodiments, the nuclease is a single-strand DNA specific nuclease. In some embodiments, the nuclease is selected from MGME1, EXOG, APEX1, APEX2, FEN1, DNA2, APE1, XRN1, XRN2, EXOG, EXOS, AP endonuclease, RecJ Exonuclease (RecJ), XseA, XseB, S1 nuclease (nucS), P1 nuclease, XRCC4, Ligase IV, Artemis, and Csml.

Except as specified above, the blunting enzymes can be from any organism. In some embodiments, the organism is a mammal. In other embodiments, the mammal is a human.

Optimal enzymes can be selected that will enable the precision indel alleles to be stably increased in various cells and target sequences. In some embodiments, the blunting enzymes can be selected from variants such as mutants, truncations or chimeric variants of DNA polymerases and single-base specific DNA nucleases. Representative variants of DNA polymerases and single-base specific DNA nucleases, including but not limited to human POLM (H329G), human POLM (H329G, R389K), human BRCT(POLM) POLL1, human BRCT(POLM) POLL2, T4 DNA polymerase(Y320A), T4 DNA polymerase(A737V). Other variants include the family X polymerases ScPolλ, HsPolλ, HsPolu, HsTdt and HsPolβ, shown schematically in FIG. 8 . In some embodiments, the blunting enzymes or the variants thereof can be modified with protein tags such as Myc, Flag, VStag, nuclease localization sequence. For example, the blunting enzymes or the variants thereof can include but not limited to 3xFlag-NLS-EXOG, 3xFlag-NLS-T4 DNA polymerase, 3xFlag-NLS-T4 DNA polymerase(Y320A), VStag-APEX2-NLS-NLS, 3xFlag-NLS-XseA.

In some embodiments, the blunting enzyme is covalently bound to the target specific nuclease by a linker. In some embodiments, the linker is an amino acid, a peptide, or a polypeptide.

Microhomology-Mediated End Joining (MMEJ) Inhibitor

The target specific nuclease and blunting enzyme disclosed herein can be combined with a microhomology-mediated end joining (MMEJ) inhibitor. In some embodiments, the MMEJ inhibitor is a CtIP inhibitor (e.g., KLHL15, PIN1). In some embodiments, the MMEJ inhibitor is an MRN inhibitor (e.g., Elb55K + E40rf6).

Pathogenic Frameshift Mutations

The non-naturally occurring or engineered systems, methods, and compositions disclosed herein can be used to repair pathogenic genes in human cells and tissues, and can be used to correct the underlying genetic basis of many diseases, especially those conditions caused by a frameshift mutation. Pathogenic frameshifts can cause a wide variety of illnesses. One particular condition caused by a frameshift mutation is Parkinson’s disease, caused by the frameshift mutation depicted in FIG. 9 .

FIG. 10 illustrates the editing of a gene using CRISPR-Cas9 and a blunting enzyme without the use of a donor template. The Cas9 gene editing system corrects a 1 bp BRCA2 frameshift mutation (c.8015_8016insA) and a 2 bp NF1 frameshift mutation (c.2027delC). As the schematic in FIG. 10 demonstrates, a stop codon is prematurely generated due to the frameshift mutation. By using this technique, combining Cas9 and a blunting enzyme without the use of a donor template results in repair of the frameshift mutations.

Other conditions caused by frameshift mutations include, inter alia, the following: various cancers, Parkinson’s disease, muscular dystrophy, cardiomyopathy, anemia, Crohn’s disease, cystic fibrosis, tuberous sclerosis, Xia-Gibbs syndrome, dermatitis, atopic, ichthyosis vulgaris, Usher syndrome, hypothyroidism, ventricular tachycardia, hemochromatosis, retinitis pigmentosa, arthrogryposis, Robinow syndrome, peroxisome biogenesis disorders, Zellweger syndrome spectrum, cortisone reductase deficiency, deficiency of pyrroline-5-carboxylate reductase, Van der Woude syndrome, Neonatal hypotonia, MYH-associated polyposis, neutropenia, methylmalonic acidemia with homocystinuria, hypobetalipoproteinemia, medium-chain acyl-coenzyme A dehydrogenase deficiency, Sezary syndrome, Stargardt disease, glycogen storage disease, maple syrup urine disease, fibrochondrogenesis, Chudley-McCullough syndrome, spastic paraplegia, frontonasal dysplasia, monocarboxylate transporter 1 deficiency, urofacial syndrome, Hajdu-Cheney syndrome, radial aplasia-thrombocytopenia syndrome, Nager syndrome, White-Sutton syndrome, ichthyosis vulgaris, FLG-Related Disorders, Grange syndrome, Charcot-Marie-Tooth disease, achromatopsia, amelogenesis imperfecta, adult junctional epidermolysis bullosa, fumarase deficiency, and Senior-Loken syndrome.

The systems, methods, and compositions described herein can also be used to enhance out-frame mutations by avoiding indel in multiples of three by a predictable mutation. Out-frame mutation occurs when the reading frame of the target dsDNA is completely disrupted. Therefore, the systems, methods, and compositions described herein can produce knockout cell lines and organisms.

Delivery

In some embodiments, the target specific nuclease and blunting enzyme are introduced into a cell as a nucleic acid encoding each protein. The nucleic acid introduced into the eukaryotic cell is a plasmid DNA or viral vector. In some embodiments, the target specific nuclease and blunting enzyme are introduced into a cell via a ribonucleoprotein (RNP).

Preferably, delivery is in the form of a vector which may be a viral vector, such as a lenti- or baculo- or adeno-viral/adeno-associated viral vectors, but other means of delivery are known (such as yeast systems, microvesicles, gene guns/means of attaching vectors to gold nanoparticles) and are provided. The viral vector may be selected from a variety of families/genera of viruses, including, but not limited to Myovindae, Siphoviridae, Podoviridae, Corticoviridae, Lipothrixviridae, Poxvirida.e, Iridoviridae, Adenoviridae, Polyomaviridae, Papillomaviridae, Mimiviridae, Pandoravirusa, Salterprovirusa, Inoviridae, Microviridae, Parvoviridae, Circoviridae, Hepadnaviridae, Caulimoviridae, Retroviridae, Cystoviridae, Reoviridae, Bimaviridae, Totiviridae, Partitivindae, Filoviridae, Orthomyxoviridae, Deltavirusa, Leviviridae, Picornavindae, Mamaviridae, Secoviridae, Poty viridae, Caliciviridae, Hepeviridae, Astroviridae, Nodaviridae, Tetraviridae, Luteoviridae, Tombusviridae, Coronaviridae, Arteriviridae, Flaviviridae, Togaviridae, Virgaviridae, Bromoviridae, Tymoviridae, Alpliallexivii-ida.e, Sobeniovirt,isa., or Idaeovirusa.

A vector may mean not only a viral or yeast system (for instance, where the nucleic acids of interest may be operably linked to and under the control of (in terms of expression, such as to ultimately provide a processed RNA) a promoter), but also direct delivery of nucleic acids into a host cell. For example, baculoviruses may be used for expression in insect cells. These insect cells may, in turn be useful for producing large quantities of further vectors, such as AAV or lentivirus adapted for delivery of the present invention. Also envisaged is a method of delivering the target specific nuclease and blunting enzyme comprising delivering to a cell mRNAs encoding each.

In some embodiments, expression of a nucleic acid sequence encoding the target specific nuclease and/or the blunting enzyme may be driven by a promoter. In some embodiments, the target specific nuclease is a Cas. In some embodiments, a single promoter drives expression of a nucleic acid sequence encoding a Cas and one or more of the guide sequences. In some embodiments, the Cas and guide sequence(s) are operably linked to and expressed from the same promoter. In some embodiments, the CRISPR enzyme and guide sequence(s) are expressed from different promoters. For example, the promoter(s) can be, but are not limited to, a UBC promoter, a PGK promoter, an EF1A promoter, a CMV promoter, an EFS promoter, a SV40 promoter, and a TRE promoter. The promoter may be a weak or a strong promoter. The promoter may be a constitutive promoter or an inducible promoter. In some embodiments, the promoter can also be an AAV ITR, and can be advantageous for eliminating the need for an additional promoter element, which can take up space in the vector. The additional space freed up by use of an AAV ITR can be used to drive the expression of additional elements, such as guide sequences. In some embodiments, the promoter may be a tissue specific promoter.

In some embodiments, an enzyme coding sequence encoding a target specific nuclease and/or a blunting enzyme is codon-optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database”, and these tables can be adapted in a number of ways. See Nakamura, Y.. et al. “codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g., 1. 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Cas protein correspond to the most frequently used codon for a particular amino acid.

In some embodiments, a vector encodes a target specific nuclease and/or a blunting enzyme comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1. 2. 3, 4, 5, 6, 7, 8, 9. 10. or more NLSs. In some embodiments, the Cas protein comprises about or more than 1, 2, 3, 4, 5, 6, 7, 8. 9. 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g., one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10. 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Typically, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, bur other types of NLS are known. In some embodiments, the NLS is between two domains, for example between the Cas 13 protein and the viral protein. The NLS may also be between two functional domains separated or flanked by a glycine-serine linker.

In general, the one or more NLSs are of sufficient strength to drive accumulation of the target specific nuclease and/or the blunting enzyme in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the target specific nuclease and/or blunting enzyme, the particular NLS used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the target specific nuclease and/or the blunting enzyme, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g.. a stain specific for the nucleus such as DAPI). Examples of detectable markers include fluorescent proteins (such as green fluorescent proteins, or GFP_(:) RFP; CFP), and epitope tags (HA tag. FLAG tag, SNAP tag). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.

In some aspects, the invention provides methods comprising delivering one or more polynucleotides, such as one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a Cas protein in combination with (and optionally complexed) with a guide sequence is delivered to a cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding a target specific nuclease and/or a blunting enzyme to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, nucleic acid complexed with a delivery vehicle, such as a liposome, and ribonucleoprotein. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-8313 (1992): Navel and Feigner, TIBTECH 11:211-217 (1993): Mitani and Caskey, TlBTECH 11:162-166 (1993); Dillon, TIBTECH 11: 167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1 149-1 154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995): Kremer and Perricaudet. British Medical Bulletin 51(1):31-44 (1995): Haddada et al., in Current Topics in Microbiology and Immunology. Doerfler and Bohm (eds) (1995): and Yu et al.. Gene Therapy 1: 13-26 (1994).

The target specific nuclease and/or the blunting enzyme can be delivered using adeno-associated virus (AAV), lentivirus, adenovirus, or other viral vector types, or combinations thereof. In some embodiments, Cas protein(s) and one or more guide RNAs can be packaged into one or more viral vectors. In some embodiments, the targeted trans-splicing system is delivered via AAV as a split intein system, similar to Levy et al. (Nature Biomedical Engineering, 2020, DOT: doi.org/10.1038/s41551-019-0501-5) in other embodiments, the target specific nuclease and/or the blunting enzyme can be delivered via AAV as a trans-splicing system, similar to Lai et al. (Nature Biotechnology, 2005, DOI: 10.1038/nbt 1153). In some embodiments, the viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the viral delivery is via intravenous, transdermal, intranasal, oral, mucosal, intrathecal, intracranial or other delivery methods. Such delivery may be either via a single dose, or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector chosen, the target cell organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc.

The use of RNA or DNA viral based systems for the delivery of nucleic acids takes advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. Viral-mediated in vivo delivery of Cas13 and guide RNA provides a rapid and powerful technology for achieving precise mRNA perturbations within cells, especially in post-mitotic cells and tissues.

In certain embodiments, delivery of the target specific nuclease and/or the blunting enzyme to a cell is non-viral. In certain embodiments, the non-viral delivery system is selected from a ribonucleoprotein, cationic lipid vehicle, electroporation, nucleofection, calcium phosphate transfection, transfection through membrane disruption using mechanical shear forces, mechanical transfection, and nanoparticle delivery.

In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g.. the American Type Culture Collection (ATCC) (Manassas, VA)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences.

Kits

The present disclosure provides kits for carrying out a method. The present disclosure provides the invention provides kits containing any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the kit comprises a vector system comprising regulatory elements and polynucleotides encoding the target specific nuclease and/or the blunting enzyme. In some embodiments, the kit comprises a viral delivery system of the target specific nuclease and/or the blunting enzyme. In some embodiments, the kit comprises a non-viral delivery system of the target specific nuclease and/or the blunting enzyme. Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube. In some embodiments, the kit includes instruction in one or more languages, for examples, in more than one language.

In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element.

Sequences

Sequences of nucleases, enzymes, guides, and linkers can be found in Table 1 below.

TABLE 1 SEQ ID NO SEQUENCE SEQ ID NO 1 atggatcccaggggtatcttgaaggcatttcccaagcggcagaaaattcatgctgatgcatcatcaaaagtacttgcaaagattcctaggagg gaagagggagaagaagcagaagagtggctgagctcccttcgggcccatgttgtgcgcactggcattggacgagcccgggcagaactctt tgagaagcagattgttcagcatggcggccagctatgccctgcccagggcccaggtgtcactcacattgtggtggatgaaggcatggactat gagcgagccctccgccttctcagactaccccagctgcccccgggtgctcagctggtgaagtcagcctggctgagcttgtgccttcaggaga ggaggctggtggatgtagctggattcagcatcttcatccccagtaggtacttggaccatccacagcccagcaaggcagagcaggatgcttc tattcctcctggcacccatgaggccctgcttcagacagccctttctcctcctcctcctcccaccaggcctgtgtctcctccccaaaaggcaaaa gaggcaccaaacacccaagcccagcccatctctgatgatgaagccagtgatggggaagaaacccaggttagtgcagctgatctggaagc POLL Homo sapiens cctcatcagtggccactaccccacctcccttgagggagattgtgagcctagcccagcccctgctgtcctggataagtgggtctgtgcacagc cctcaagccagaaggcgaccaatcacaacctccatatcacagagaagctggaagttctggccaaagcctacagtgttcagggagacaagt ggagggccctgggctatgccaaggccatcaatgccctcaagagcttccataagcctgtcacctcgtaccaggaggcctgcagtatccctg ggattgggaagcggatggctgagaaaatcatagagatcctggagagcgggcatttgcggaagctggaccatatcagtgagagcgtgcct gtcttggagctcttctccaacatctggggagctgggaccaagactgcccagatgtggtaccaacagggcttccgaagtctggaagacatcc gcagccaggcctccctgacaacccagcaggccatcggcctgaagcattacagtgacttcctggaacgtatgcccagggaggaggctaca gagattgagcagacagtccagaaagcagcccaggcctttaactctgggctgctgtgtgtggcatgtggttcataccgacggggaaaggcg acctgtggtgatgtcgacgtgctcatcactcacccagatggccggtcccaccggggtatcttcagccgcctccttgacagtcttcggcagga agggttcctcacagatgacttggtgagccaagaggagaatggtcagcaacagaagtacttgggggtgtgccggctcccagggccagggc ggcggcaccggcgcctggacatcatcgtggtgccctatagcgagtttgcctgtgccctgctctacttcaccggctctgcacacttcaaccgc tccatgcgagccctggccaaaaccaagggcatgagtctgtcagaacatgccctcagcactgctgtggtccggaacacccatggctgcaag gtggggcctggccgagtgctgcccactcccactgagaaggatgtcttcaggctcttaggcctcccctaccgagaacctgctgagcgggac tggtga SEQ ID NO 2 atggctctgccaaagagaagacgcgcacgggttgggtccccttcaggggacgcagcctcctctacacctccatctacgagatttccgggtgtt gcaatatacctcgtcgagccccggatgggacgcagtagacgggctttccttacgggtctcgcccgaagtaaaggctttcgggtgttggacgca tgttctagtgaagcgacccatgtc gttatggaggagac gagtgctgaggaggctgtaagctggcaagagcgccgaatggctgctgcaccacc cggctgcactccgcctgctttgttggatatatcttggctgaccgaaagtcttggggctggacaaccagtaccggttgagtgccgacatcgattgg aagtagctggtccgaggaaaggccccctctcaccggcctggatgcctgcatatgcctgccaacggcccacccctctgacgcaccacaacact gggctttccgaggcattggagatattggcggaagctgcgggctttgaagggtccgaagggagattgctcacgttctgtagagcagcatccgttc ttaaagcgctgccgagtcccgtaactacactgtctcaactgcagggcctgccacacttcggcgaacactcaagccgggtcgtacaagaactcc tggagcacggggtctgcgaggaagttgagagggtgaggcgaagcgaacgataccaaacgatgaagctgtttacacaaatctttggagttgga gtcaagacggcggacagatggtatcgagaggggcttcgaacgctcgacgatctgcgcgagcaaccgcaaaagctgacccaacagcaaaa ggccggactgcagcatcaccaggacctttcaacacctgttcttcggtctgacgttgatgctctccaacaagtcgtcgaggaggcagtaggccag gcccttccgggcgctactgttacgctcacgggaggatttagacgcggcaaacttcaaggtcacgatgtcgatttcttgataactcacccaaaaga ggggcaggaggctggtttgctgccgcgggtaatgtgccgattgcaagaccaaggcttgatactgtaccaccaacaccaacattcatgttgcga gtcacccacgcgcctcgcacagcagagccatatggacgctttcgagagatcattttgcatattcagacttcctcagcccccaggtgcggcggtc ggtgggtccactaggccgtgtccatcttggaaggctgtgcgggtggatttggtcgtagcgcccgtcagccagtttccctttgcactcctggggtg gaccggcagtaaactgttccaaagagagctgcgaaggttctcacgaaaagagaagggcctctggcttaactcccacggcctgttcgaccccg agcaaaaaactttctttcaggctgcgagcgaagaagatatcttccgccacctgggacttgagtaccttccccccgagcagcgcaacgcctga POLM Homo sapiens SEQ ID NO 3 atggctctgccaaagagaagacgcgcacgggttgggtccccttcaggggacgcagcctcctctacacctccatctacgagatttccgggtgtt gcaatatacctcgtcgagccccggatgggacgcagtagacgggctttccttacgggtctcgcccgaagtaaaggctttcgggtgttggacgca tgttctagtgaagcgacccatgtc gttatggaggagac gagtgctgaggaggctgtaagctggcaagagcgccgaatggctgctgcaccacc cggctgcactccgcctgctttgttggatatatcttggctgaccgaaagtcttggggctggacaaccagtaccggttgagtgccgacatcgattgg aagtagctggtccgaggaaaggccccctctcaccggcctggatgcctgcatatgcctgccaacggcccacccctctgacgcaccacaacact gggctttccgaggcattggagatattggcggaagctgcgggctttgaagggtccgaagggagattgctcacgttctgtagagcagcatccgttc ttaaagcgctgccgagtcccgtaactacactgtctcaactgcagggcctgccacacttcggcgaacactcaagccgggtcgtacaagaactcc tggagcacggggtctgcgaggaagttgagagggtgaggcgaagcgaacgataccaaacgatgaagctgtttacacaaatctttggagttgga gtcaagacggcggacagatggtatcgagaggggcttcgaacgctcgacgatctgcgcgagcaaccgcaaaagctgacccaacagcaaaa ggccggactgcagcatcaccaggacctttcaacacctgttcttcggtctgacgttgatgctctccaacaagtcgtcgaggaggcagtaggccag gcccttccgggcgctactgttacgctcacgggaggatttagacgcggcaaacttcaaggtggcgatgtcgatttcttgataactcacccaaaag aggggcaggaggctggtttgctgccgcgggtaatgtgccgattgcaagaccaaggcttgatactgtaccaccaacaccaacattcatgttgcg agtcacccacgcgcctcgcacagcagagccatatggacgctttcgagagatcaaaatgcatattcagacttcctcagcccccaggtgcggcg gtcggtgggtccactaggccgtgtccatcttggaaggctgtgcgggtggatttggtcgtagcgcccgtcagccagtttccctttgcactcctggg gtggaccggcagtaaactgttccaaagagagctgcgaaggttctcacgaaaagagaagggcctctggcttaactcccacggcctgttcgacc ccgagcaaaaaactttctttcaggctgcgagcgaagaagatatcttccgccacctgggacttgagtaccttccccccgagcagcgcaacgcct ga POLM(H329 G) Homo sapiens SEQ ID NO 4 atggctctgccaaagagaagacgcgcacgggttgggtccccttcaggggacgcagcctcctctacacctccatctacgagatttccgggtgtt gcaatatacctcgtcgagccccggatgggacgcagtagacgggctttccttacgggtctcgcccgaagtaaaggctttcgggtgttggacgca tgttctagtgaagcgacccatgtc gttatggaggagac gagtgctgaggaggctgtaagctggcaagagcgccgaatggctgctgcaccacc cggctgcactccgcctgctttgttggatatatcttggctgaccgaaagtcttggggctggacaaccagtaccggttgagtgccgacatcgattgg aagtagctggtccgaggaaaggccccctctcaccggcctggatgcctgcatatgcctgccaacggcccacccctctgacgcaccacaacact gggctttccgaggcattggagatattggcggaagctgcgggctttgaagggtccgaagggagattgctcacgttctgtagagcagcatccgttc ttaaagcgctgccgagtcccgtaactacactgtctcaactgcagggcctgccacacttcggcgaacactcaagccgggtcgtacaagaactcc tggagcacggggtctgcgaggaagttgagagggtgaggcgaagcgaacgataccaaacgatgaagctgtttacacaaatctttggagttgga gtcaagacggcggacagatggtatcgagaggggcttcgaacgctcgacgatctgcgcgagcaaccgcaaaagctgacccaacagcaaaa ggccggactgcagcatcaccaggacctttcaacacctgttcttcggtctgacgttgatgctctccaacaagtcgtcgaggaggcagtaggccag POLM(H329 G,R389K) Homo sapiens gcccttccgggcgctactgttacgctcacgggaggatttagacgcggcaaacttcaaggtggcgatgtcgatttcttgataactcacccaaaag aggggcaggaggctggtttgctgccgcgggtaatgtgccgattgcaagaccaaggcttgatactgtaccaccaacaccaacattcatgttgcg agtcacccacgcgcctcgcacagcagagccatatggacgctttcgagagatcattttgcatattcagacttcctcagcccccaggtgcggcggt cggtgggtccactaggccgtgtccatcttggaaggctgtgcgggtggatttggtcgtagcgcccgtcagccagtttccctttgcactcctggggt ggaccggcagtaaactgttccaaagagagctgcgaaggttctcacgaaaagagaagggcctctggcttaactcccacggcctgttcgacccc gagcaaaaaactttctttcaggctgcgagcgaagaagatatcttccgccacctgggacttgagtaccttccccccgagcagcgcaacgcctga SEQ ID NO 5 atggctctgccaaagagaagacgcgcacgggttgggtccccttcaggggacgcagcctcctctacacctccatctacgagatttccgggtgtt gcaatatacctcgtcgagccccggatgggacgcagtagacgggctttccttacgggtctcgcccgaagtaaaggctttcgggtgttggacgca tgttctagtgaagcgacccatgtcgttatggaggagacgagtgctgaggaggctgtaagctggcaagagcgccgaatggctgctgcaccacc cggctgcactccgcctgctttgttggatatatcttggctgaccgaaagtcttggggctggacaaccaatccccagtaggtacttggaccatccac agcccagcaaggcagagcaggatgcttctattcctcctggcacccatgaggccctgcttcagacagccctttctcctcctcctcctcccaccag gcctgtgtctcctccccaaaaggcaaaagaggcaccaaacacccaagcccagcccatctctgatgatgaagccagtgatggggaagaaacc caggttagtgcagctgatctggaagccctcatcagtggccactaccccacctcccttgagggagattgtgagcctagcccagcccctgctgtcc tggataagtgggtctgtgcacagccctcaagccagaaggcgaccaatcacaacctccatatcacagagaagctggaagttctggccaaagcc tacagtgttcagggagacaagtggagggccctgggctatgccaaggccatcaatgccctcaagagcttccataagcctgtcacctcgtaccag gaggcctgcagtatccctgggattgggaagcggatggctgagaaaatcatagagatcctggagagcgggcatttgcggaagctggaccatat cagtgagagcgtgcctgtcttggagctcttctccaacatctggggagctgggaccaagactgcccagatgtggtaccaacagggcttccgaag tctggaagacatccgcagccaggcctccctgacaacccagcaggccatcggcctgaagcattacagtgacttcctggaacgtatgcccaggg aggaggctacagagattgagcagacagtccagaaagcagcccaggcctttaactctgggctgctgtgtgtggcatgtggttcataccgacggg gaaaggcgacctgtggtgatgtcgacgtgctcatcactcacccagatggccggtcccaccggggtatcttcagccgcctccttgacagtcttcg gcaggaagggttcctcacagatgacttggtgagccaagaggagaatggtcagcaacagaagtacttgggggtgtgccggctcccagggcca gggcggcggcaccggcgcctggacatcatcgtggtgccctatagcgagtttgcctgtgccctgctctacttcaccggctctgcacacttcaacc gctccatgcgagccctggccaaaaccaagggcatgagtctgtcagaacatgccctcagcactgctgtggtccggaacacccatggctgcaag gtggggcctggccgagtgctgcccactcccactgagaaggatgtcttcaggctcttaggcctcccctaccgagaacctgctgagcgggactg gtga BRCT(POLM ) POLL 1 Homo sapiens SEQ ID NO 6 atggctctgccaaagagaagacgcgcacgggttgggtccccttcaggggacgcagcctcctctacacctccatctacgagatttccgggtgtt gcaatatacctcgtcgagccccggatgggacgcagtagacgggctttccttacgggtctcgcccgaagtaaaggctttcgggtgttggacgca tgttctagtgaagcgacccatgtc gttatggaggagac gagtgctgaggaggctgtaagctggcaagagcgccgaatggctgctgcaccacc cggctgcactccgcctgctttgttggatatatcttggctgaccgaaagtcttggggctggacaaccagtaccggttgagtgccgacatcgattgg aagtagctggtccgaggaaaggccccctctcatcaagccagaaggcgaccaatcacaacctccatatcacagagaagctggaagttctggcc aaagcctacagtgttcagggagacaagtggagggcccigggvidigvcaaggccatcaatgccctcaagagcttccataagcctgtcaccteg taccaggaggcctgcagtatccctgggattgggaagcggatggctgagaaaatcatagagatcctggagagcgggcatttgcggaagctgg accatatcagtgagagcgtgcctgtcttggagctcttctccaacatctggggagctgggaccaagactgcccagatgtggtaccaacagggctt ccgaagtctggaagacatccgcagccaggcctccctgacaacccagcaggccatcggcctgaagcattacagtgacttcctggaacgtatgc ccagggaggaggctacagagattgagcagacagtccagaaagcagcccaggcctttaactctgggctgctgtgtgtggcatgtggttcatacc gacggggaaaggcgacctgtggtgatgtcgacgtgctcatcactcacccagatggccggtcccaccggggtatcttcagccgcctccttgaca gtcttcggcaggaagggttcctcacagatgacttggtgagccaagaggagaatggtcagcaacagaagtacttgggggtgtgccggctccca gggccagggcggcggcaccggcgcctggacatcatcgtggtgccctatagcgagtttgcctgtgccctgctctacttcaccggctctgcacac ttcaaccgctccatgcgagccctggccaaaaccaagggcatgagtctgtcagaacatgccctcagcactgctgtggtccggaacacccatgg ctgcaaggtggggcctggccgagtgctgcccactcccactgagaaggatgtcttcaggctcttaggcctcccctaccgagaacctgctgagcg ggactggtga BRCT(POLM )_POLL2 Homo sapiens SEQ ID NO 7 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgctatcaagagtatcgcttcccgcctccggggttcccgtcgttttctgagcggcttcgtggctggg gctgtagtgggcgctgcgggagctgggctcgcggccctgcagttcttccggagtcagggcgctgagggagcgttgacagggaagcagccg gatggatctgcagaaaaggctgtcttggaacaatttggattccctttaactggaacagaggcaaggtgttacactaatcacgctttgtcttatgatc aggcaaagcgggtgcctagatgggttcttgaacatatttccaaaagcaagataatgggtgatgcagacagaaagcattgtaaatttaagcctgat cccaatatccctccaaccttcagtgccttcaatgaagattatgttggaagtgggtggtcacgaggacacatggctccagcaggaaataacaaatt ttcaagtaaagccatggctgaaaccttttacctttctaacattgtgcctcaggattttgataataattctggatattggaacagaatagaaatgtactgt cgagagctgacagaaaggtttgaagatgtttgggtggtatctgggcctttgaccttacctcagactagaggcgatggaaagaaaatagttagtta ccaggtgattggcgaggacaacgtggcagtcccctcacacctttataaggtaatcctggcccgcagaagctcagtatctaccgaaccactggc gctaggggcctttgtggtacccaatgaagccatcggcttccagccccagttaactgaattccaagtgagcctccaggacctagagaagttgtca ggactggtgttttttcctcatttggatagaactagtgatatccggaatatctgctctgtggacacctgtaagctcctggatttccaggagttcaccttgt acttgagtacaagaaagattgaaggagcccgatcagtgctcagactggaaaagatcatggaaaacttgaagaatgcagagattgaaccagatg attactttatgagtcgctatgagaagaagctagaagaactcaaagctaaggagcagtcaggaacccagataagaaagccatcctag 3xFlag-NLS-EXOG Homo sapiens SEQ ID NO 8 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagcccccaggctcctgcctatatccgccgcaaccctcgctctggcccaacttacttatggctggggcaat 3xFlag-NLS-nucS ctgggccatgaaactgtcgcttacattgctcaatctttcgtcgcgtcaagtaccgagagcttctgccaaaacatattgggggacgactctacttcat atttggccaacgtggcaacatgggcggatacttacaaatatacggatgcgggcgaatttagcaaaccctatcactttatagacgcacaggataa cccaccccaatcatgcggggttgactatgacagggattgtgggtccgccgggtgctctatctcagcaattcaaaactacacgaacatactgctg gaaagtcctaatgggagcgaggctctgaacgcactgaaatttgttgtccatattataggagatattcatcagccgttgcatgacgaaaatttggag gcaggaggaaatggcatcgatgtgacatatgacggggagactacgaaccttcatcacatttgggatactaacatgccggaagaagccgcggg agggtatagcttgtccgtggcaaagacttatgcagatttgctcaccgagaggataaaaacaggtacttactcctcaaaaaaggatagctggacc gatggaattgatataaaagatccagtaagcacgtctatgatttgggcggcggatgcaaacacctacgtctgtagtacggtacttgatgacggtctt gcttatattaattccactgacctctccggcgaatactacgacaagtcacaaccagtcttcgaagaacttatagccaaagcgggttatagacttgcg gcttggctggaccttattgcgtcccagcccagctga Aspergillus oryzae SEQ ID NO 9 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagcctggggtgccctcggccatgcgacagtagcctatgtagctcaacattatgtaagtcccgaagccgc gtcttgggcgcaaggcattttgggttcctcaagttcatcatatttggcttcaatagcttcttgggccgatgaataccggctgacctccgccggcaag tggagtgctagtttgcactttattgatgccgaagataatccacccacgaactgcaacgtcgactatgaacgggattgtggatcttccgggtgctcc atatcagctatagctaattatacacagcgagtaagtgactcaagtctttcttccgaaaatcatgcggaagcactgcgattcttggtacacttcatcgg ggacatgacacagcctttgcacgatgaagcctacgcggtgggcggtaataaaataaacgttacatttgatggttatcatgacaacctgcacagc gattgggacacgtatatgccacagaaattgatcggcggtcatgcgctttcagacgcagagtcctgggcaaagacgctggttcaaaatatcgaat ctggaaattacaccgcgcaggccattggttggatcaaaggcgacaacatctcagaaccaatcacaaccgcaacgcgatgggcgtcagacgc caatgctcttgtatgtacggtggttatgcctcacggagctgcggcacttcagacaggtgacctttatccgacttactacgactctgtgatagatact attgaacttcaaatagctaaaggaggctaccggctcgcgaactggataaacgagatatag 3xFlag-NLS-NucPl Penicillium citrinum SEQ ID NO 10 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccaagatgaagttatttcagaccatttgcaggcagctcaggagttcaaagttttctgtggaatcagctg cccttgtggctttctctacttcctcttactcatgtggccggaagaaaaaagtgaacccatatgaagaagtggaccaagaaaaatactctaatttagtt cagtctgtcttgtcatccagaggcgtcgcccagaccccgggatcggtggaggaagatgctttgctctgtggacccgtgagcaagcataagctg ccaaaccaaggtgaggacagacgagtgccacaaaactggtttcctatcttcaatccagagagaagtgataaaccaaatgcaagtgatccttcag ttcctttgaaaatccccttgcaaaggaatgtgataccaagtgtgacccgagtccttcagcagaccatgacaaaacaacaggttttcttgttggaga ggtggaaacagcggatgattctggaactgggagaagatggctttaaagaatacacttcaagttttcatgtttgtgatcatgtgtatatgaagaacct agccagggacgtctttttacaagggaaacggttccacgaagccttggaaagcatactttcaccccaggaaaccttaaaagagagagatgaaaa tctcctcaagtctggttacattgaaagtgtccagcatattctgaaagatgtcagtggagtgcgagctcttgaaagtgctgttcaacatgaaaccttaa actatataggtctgctggactgtgtggctgagtatcagggcaagctctgtgtgattgattggaagacatcagagaaaccaaagccttttattcaaa gtacatttgacaacccactgcaagttgtggcatacatgggtgccatgaaccatgataccaactacagctttcaggttcaatgtggcttaattgtggt ggcctacaaagatggatcacctgcccacccacatttcatggatgcagagctctgttcccagtactggaccaagtggcttcttcgactagaagaat atacggaaaagaaaaagaaccagaatattcagaaaccagaatattcagaatag 3xFlag-NLS-MGME1 Homo sapiens SEQ ID NO 11 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgtgaagcagcagatccaacttagacgaagggaagtcgacgaaacagcggaccttccggctga gttgcctccccttcttagac gattgtatgcaagtcgcggggttcgctctgcacaggaacttgagcgctctgtcaagggaatgctgccctggcaac agttgagtggtgttgaaaaggccgttgagattctctataatgcattcagggaaggaactcggatcatagtggtaggtgatttcgacgctgatgga gcaacttcaaccgcgttgagcgtactcgccatgcgctctctcgggtgctcaaacattgactatttggtccccaatcgatttgaagatggttatggac tcagcccggaggtggttgaccaagcgcatgcccggggcgcccagctcatcgtcactgtcgataacgggataagctctcacgccggtgtcgaa cacgctcgcagcctcgggattcccgtgattgtgactgaccaccaccttccgggagatacactccccgctgctgaggcaataatcaatcctaacc ttcgggattgtaactttccgagcaaatcactcgcaggggtaggggtcgcattctatctcatgctggcgctcagaacgttccttcgagatcaggggt ggttcgacgagcggaacatagctatacctaatttggccgaacttttggatctcgtggcgcttggcacggttgcagacgttgtccctctggacgcg aacaatcgaattttgacatggcagggaatgtctaggattagagccgggaaatgtaggcctggtattaaagctctcttggaggtggcaaaccgag atgcccagaagctcgcagctagtgacttgggttttgctttgggaccccgcctgaacgctgcagggcgcctggatgacatgagcgtaggcgtag cacttctcttgtgcgacaatataggtgaagcgagagtacttgcaaacgaactggatgcgcttaaccagacaagaaaggaaattgagcagggca tgcagatagaggcgcttaccctgtgtgaaaagctcgaacgatctcgagacacccttccaggcggactcgcgatgtatcaccctgaatggcacc agggtgtcgtaggcatcctcgcgtcccgcataaaagaaaggttccaccggccagttatagcttttgctcccgcaggtgatggaacccttaaagg atctgggagatctatccaggggcttcatatgcgggatgctttggagcggcttgacactctttacccaggtatgatgctcaagttcggcggtcatgc tatggctgccggcctctcactggaggaggataaatttaaactctttcaacagaggttcggggagcttgtgacggaatggctggatccgtccttgc ttcaaggcgaagtagttagcgacggacctctcagtcctgcggagatgacgatggaggtagcgcaactgctcagggacgctgggccgtgggg ccagatgttcccggagccgttgttcgacggccatttcaggttgcttcaacagcgcctcgtcggagaacggcatctcaaagtaatggttgagcca gtcggtggcggccccctgcttgatggcatcgctttcaatgtagacactgcactgtggcccgataatggcgttcgagaggttcagcttgcctataa gctggatattaacgagtttcgagggaaccgatctctgcaaattataatagacaatatctggcccatatag 3xFlag-NLS-RecJ Escherichia coli SEQ ID NO 12 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccaaggaattctatattagtatcgagacagtgggaaacaacattgtagagcggtatatcgatgaaaac ggcaaagaaaggactcgagaggtcgaataccttccgaccatgtttcgccactgtaaagaagaatctaagtacaaagatatttacgggaagaatt gcgctccccaaaaatttccctccatgaaggatgctcgagactggatgaagcgcatggaggacataggtctcgaagcattggggatgaacgatt SEQ ID NO SEQUENCE 3xFlag-NLS-T4 DNA polymerase ttaagttggcttacatctccgacacttacgggtcagaaatagtgtacgataggaagttcgttcgcgtggcaaattgcgatatagaggtcactggag ataagttcccggacccgatgaaggcggagtacgaaattgacgctataacacactatgactcaatcgacgaccggttctatgtatttgacctgctc aattccatgtacgggtctgtaagcaagtgggacgctaaactcgcggctaaacttgactgtgaaggaggggatgaagtacctcaggaaatcttgg acagggtaatctacatgccctttgacaatgaacgagatatgcttatggagtacattaatttgtgggagcaaaagcgccccgcaatatttacaggct ggaacatagaagggttc gatgtaccgtatattatgaatcgggtaaagatgatcctcggagagagaagcatgaaaagattttcacctattggcaga gtgaaatctaagttgatacaaaacatgtatggctcaaaagagatctattcaatagatggagttagcatactcgactacctggatctgtataaaaagtt tgcttttaccaacttgcctagcttctcccttgaaagtgtcgcccaacacgagaccaagaaaggtaagctgccgtacgatggcccgattaataaact gcgcgagaccaaccatcaaagatatattagctacaacataattgatgtcgaatctgtgcaagccattgataagataaggggctttatcgaccttgt cctgtcaatgtcctattacgccaaaatgccgttctcaggtgtaatgtcacccataaagacgtgggatgcgatcatcttcaattctctcaagggaga gcacaaggtgatcccccaacaggggtcccacgtaaagcagtccttcccgggagcttttgtctttgagccaaagccgatcgcccgaaggtatat catgtctttcgaccttacgtcactttacccttcaattattcgacaagtgaatatatcacccgagactatccggggccagtttaaggtacacccaatcc atgagtacatagccggtacagccccaaaacccagtgacgaatactcttgcagccctaatgggtggatgtacgacaaacaccaggagggcata atcccaaaggaaatcgcgaaagtamttccaacggaaagactggaagaaaaagatgttcgcggaggaaatgaacgccgaggctattaaaaa aatcattatgaagggagcgggtagctgttctaccaagccagaggtagagcgctacgtcaaattcagtgatgacttccttaatgagctgagtaact acacagagtctgtactgaactcactgattgaggaatgtgaaaaagccgcaacacttgctaataccaatcaactgaatcggaagatcctgattaatt cactgtatggcgccttgggcaacattcatttcagatactacgacctcaggaatgccacggccattacaattttcggtcaggtcgggatccagtgg atcgcccgaaaaatcaacgagtacctcaataaagtgtgtggtaccaatgacgaggattttatcgcagcaggcgataccgatagcgtgtatgtttg cgtcgacaaggtcattgaaaaggtagggctggatcggtttaaggagcagaatgatcttgtcgagtttatgaaccagtttggtaaaaaaaagatgg aaccgatgatagatgtagcgtaccgagaactttgtgactacatgaataatcgcgagcacttgatgcacatggacagggaagcgatttcatgccc cccactcggttcaaagggcgtagggggtttctggaaagctaagaaacggtacgccctcaacgtctatgacatggaagacaagaggttcgcgg aacctcatttgaagataatggggatggagacgcaacagtcctcaactccaaaggctgtgcaagaggctctggaagaaagcatacgacgcata ctccaggagggggaagagagtgttcaggagtattataaaaactttgaaaaggagtaccgccagcttgactacaagghaatcgcggaggttaag actgcgaatgatatcgccaaatatgatgataaaggatggcccggtttcaaatgccctttccatatacgaggggtcctcacctaccgccgcgccgt gtctggtctgggggtcgcaccaattctcgacggaaataaggttatggtactcccactccgcgaggggaatccgtttggtgacaaatgcatcgcc tggccgtctggtacggagctccccaaggaaatacgcagcgacgtcctcagttggatcgaccactccacactgtttcagaagtcattcgttaaac ctctggccgggatgtgtgaatccgcgggtatggactacgaggagaaagcttcattggactttcttttcggttga Bacteriophage t4 SEQ ID NO 13 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccaaggaattctatattagtatcgagacagtgggaaacaacattgtagagcggtatatcgatgaaaac ggcaaagaaaggactcgagaggtcgaataccttccgaccatgtttcgccactgtaaagaagaatctaagtacaaagatatttacgggaagaatt gcgctccccaaaaatttccctccatgaaggatgctcgagactggatgaagcgcatggaggacataggtctcgaagcattggggatgaacgatt ttaagttggcttacatctccgacacttacgggtcagaaatagtgtacgataggaagttcgttcgcgtggcaaattgcgatatagaggtcactggag ataagttcccggacccgatgaaggcggagtacgaaattgacgctataacacactatgactcaatcgacgaccggttctatgtatttgacctgctc aattccatgtacgggtctgtaagcaagtgggacgctaaactcgcggctaaacttgactgtgaaggaggggatgaagtacctcaggaaatcttgg acagggtaatctacatgccctttgacaatgaacgagatatgcttatggagtacattaatttgtgggagcaaaagcgccccgcaatatttacaggct ggaacatagaagggttc gatgtaccgtatattatgaatcgggtaaagatgatcctcggagagagaagcatgaaaagattttcacctattggcaga gtgaaatctaagttgatacaaaacatgtatggctcaaaagagatctattcaatagatggagttagcatactcgactacctggatctgtataaaaagtt tgcttttaccaacttgcctagcttctcccttgaaagtgtcgcccaacacgagaccaagaaaggtaagctgccgtacgatggcccgattaataaact gcgcgagaccaaccatcaaagatatattagcgccaacataattgatgtcgaatctgtgcaagccattgataagataaggggctttatcgaccttgt cctgtcaatgtcctattacgccaaaatgccgttctcaggtgtaatgtcacccataaagacgtgggatgcgatcatcttcaattctctcaagggaga gcacaaggtgatcccccaacaggggtcccacgtaaagcagtccttcccgggagcttttgtctttgagccaaagccgatcgcccgaaggtatat catgtctttcgaccttacgtcactttacccttcaattattcgacaagtgaatatatcacccgagactatccggggccagtttaaggtacacccaatcc atgagtacatagccggtacagccccaaaacccagtgacgaatactcttgcagccctaatgggtggatgtacgacaaacaccaggagggcata atcccaaaggaaatcgcgaaagtamttccaacggaaagactggaagaaaaagatgttcgcggaggaaatgaacgccgaggctattaaaaa aatcattatgaagggagcgggtagctgttctaccaagccagaggtagagcgctacgtcaaattcagtgatgacttccttaatgagctgagtaact acacagagtctgtactgaactcactgattgaggaatgtgaaaaagccgcaacacttgctaataccaatcaactgaatcggaagatcctgattaatt cactgtatggcgccttgggcaacattcatttcagatactacgacctcaggaatgccacggccattacaattttcggtcaggtcgggatccagtgg atcgcccgaaaaatcaacgagtacctcaataaagtgtgtggtaccaatgacgaggattttatcgcagcaggcgataccgatagcgtgtatgtttg cgtcgacaaggtcattgaaaaggtagggctggatcggtttaaggagcagaatgatcttgtcgagtttatgaaccagtttggtaaaaaaaagatgg aaccgatgatagatgtagcgtaccgagaactttgtgactacatgaataatcgcgagcacttgatgcacatggacagggaagcgatttcatgccc cccactcggttcaaagggcgtagggggtttctggaaagctaagaaacggtacgccctcaacgtctatgacatggaagacaagaggttcgcgg aacctcatttgaagataatggggatggagacgcaacagtcctcaactccaaaggctgtgcaagaggctctggaagaaagcatacgacgcata ctccaggagggggaagagagtgttcaggagtattataaaaactttgaaaaggagtaccgccagcttgactacaagghaatcgcggaggttaag actgcgaatgatatcgccaaatatgatgataaaggatggcccggtttcaaatgccctttccatatacgaggggtcctcacctaccgccgcgccgt gtctggtctgggggtcgcaccaattctcgacggaaataaggttatggtactcccactccgcgaggggaatccgtttggtgacaaatgcatcgcc tggccgtctggtacggagctccccaaggaaatacgcagcgacgtcctcagttggatcgaccactccacactgtttcagaagtcattcgttaaac ctctggccgggatgtgtgaatccgcgggtatggactacgaggagaaagcttcattggactttcttttcggttga 3xFlag-NLS-T4 DNA polymerase(Y 320A) Bacteriophage t4 SEQ ID NO 14 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccaaggaattctatattagtatcgagacagtgggaaacaacattgtagagcggtatatcgatgaaaac ggcaaagaaaggactcgagaggtcgaataccttccgaccatgtttcgccactgtaaagaagaatctaagtacaaagatatttacgggaagaatt gcgctccccaaaaatttccctccatgaaggatgctcgagactggatgaagcgcatggaggacataggtctcgaagcattggggatgaacgatt ttaagttggcttacatctccgacacttacgggtcagaaatagtgtacgataggaagttcgttcgcgtggcaaattgcgatatagaggtcactggag ataagttcccggacccgatgaaggcggagtacgaaattgacgctataacacactatgactcaatcgacgaccggttctatgtatttgacctgctc aattccatgtacgggtctgtaagcaagtgggacgctaaactcgcggctaaacttgactgtgaaggaggggatgaagtacctcaggaaatcttgg acagggtaatctacatgccctttgacaatgaacgagatatgcttatggagtacattaatttgtgggagcaaaagcgccccgcaatatttacaggct ggaacatagaagggttcgatgtaccgtatattatgaatcgggtaaagatgatcctcggagagagaagcatgaaaagattttcacctattggcaga gtgaaatctaagttgatacaaaacatgtatggctcaaaagagatctattcaatagatggagttagcatactcgactacctggatctgtataaaaagtt tgcttttaccaacttgcctagcttctcccttgaaagtgtcgcccaacacgagaccaagaaaggtaagctgccgtacgatggcccgattaataaact gcgcgagaccaaccatcaaagatatattagctacaacataattgatgtcgaatctgtgcaagccattgataagataaggggctttatcgaccttgt cctgtcaatgtcctattacgccaaaatgccgttctcaggtgtaatgtcacccataaagacgtgggatgcgatcatcttcaattctctcaagggaga gcacaaggtgatcccccaacaggggtcccacgtaaagcagtccttcccgggagcttttgtctttgagccaaagccgatcgcccgaaggtatat catgtctttcgaccttacgtcactttacccttcaattattcgacaagtgaatatatcacccgagactatccggggccagtttaaggtacacccaatcc atgagtacatagccggtacagccccaaaacccagtgacgaatactcttgcagccctaatgggtggatgtacgacaaacaccaggagggcata atcccaaaggaaatcgcgaaagtamttccaacggaaagactggaagaaaaagatgttcgcggaggaaatgaacgccgaggctattaaaaa aatcattatgaagggagcgggtagctgttctaccaagccagaggtagagcgctacgtcaaattcagtgatgacttccttaatgagctgagtaact acacagagtctgtactgaactcactgattgaggaatgtgaaaaagccgcaacacttgctaataccaatcaactgaatcggaagatcctgattaatt cactgtatggcgccttgggcaacattcatttcagatactacgacctcaggaatgccacggccattacaattttcggtcaggtcgggatccagtgg atcgcccgaaaaatcaacgagtacctcaataaagtgtgtggtaccaatgacgaggattttatcgcagcaggcgataccgatagcgtgtatgtttg cgtcgacaaggtcattgaaaaggtagggctggatcggtttaaggagcagaatgatcttgtcgagtttatgaaccagtttggtaaaaaaaagatgg aaccgatgatagatgtagcgtaccgagaactttgtgactacatgaataatcgcgagcacttgatgcacatggacagggaagcgatttcatgccc cccactcggttcaaagggcgtagggggtttctggaaagctaagaaacggtacgccctcaacgtctatgacatggaagacaagaggttcgcgg aacctcatttgaagataatggggatggagacgcaacagtcctcaactccaaaggtggtgcaagaggctctggaagaaagcatacgacgcata ctccaggagggggaagagagtgttcaggagtattataaaaactttgaaaaggagtaccgccagcttgactacaagghaatcgcggaggttaag actgcgaatgatatcgccaaatatgatgataaaggatggcccggtttcaaatgccctttccatatacgaggggtcctcacctaccgccgcgccgt gtctggtctgggggtcgcaccaattctcgacggaaataaggttatggtactcccactccgcgaggggaatccgtttggtgacaaatgcatcgcc tggccgtctggtacggagctccccaaggaaatacgcagcgacgtcctcagttggatcgaccactccacactgtttcagaagtcattcgttaaac ctctggccgggatgtgtgaatccgcgggtatggactacgaggagaaagcttcattggactttcttttcggttga 3xFlag-NLS-T4 DNA polymerase(A 737V) Bacteriophage t4 SEQ ID NO 15 atggctccgaagcgtgggaaaaagggagcggtggcggaagacggggatgagctcaggacagagccagaggccaagaagagtaagacg gccgcaaagaaaaatgacaaagaggcagcaggagagggcccagccctgtatgaggaccccccagatcagaaaacctcacccagtggcaa acctgccacactcaagatctgctcttggaatgtggatgggcttcgagcctggattaagaagaaaggattagattgggtaaaggaagaagcccc agatatactgtgccttcaagagaccaaatgttcagagaacaaactaccagctgaacttcaggagctgcctggactctctcatcaatactggtcag ctccttcggacaaggaagggtacagtggcgtgggcctgctttcccgccagtgcccactcaaagtttcttacggcataggcgatgaggagcatg atcaggaaggccgggtgattgtggctgaatttgactcgtttgtgctggtaacagcatatgtacctaatgcaggccgaggtctggtacgactggag taccggcagcgctgggatgaagcctttcgcaagttcctgaagggcctggcttcccgaaagccccttgtgctgtgtggagacctcaatgtggcac atgaagaaattgaccttcgcaaccccaaggggaacaaaaagaatgctggcttcacgccacaagagcgccaaggcttcggggaattactgcag gctgtgccactggctgacagctttaggcacctctaccccaacacaccctatgcctacaccttttggacttatatgatgaatgctc gatccaagaatg ttggttggcgccttgattactttttgttgtcccactctctgttacctgcattgtgtgacagcaagatccgttccaaggccctcggcagtgatcactgtc ctatcaccctatacctagcactgtga APEX1 Homo sapiens SEQ ID NO 16 atggtgcggggttctggcaagcccatccccaaccccctgctgggcctggacagcaccggaaagtcttacccaactgtgagtgctgattaccag gacgccgttgagaaggcgaagaagaagctcagaggcttcatcgctgagaagagatgcgctcctctaatgctccgtttggcattccactctgctg gaacctttgacaagggcacgaagaccggtggacccttcggaaccatcaagcaccctgccgaactggctcacagcgctaacaacggtcttgac atcgctgttaggcttttggagccactcaaggcggagttccctattttgagctacgccgatttctaccagttggctggcgttgttgccgttgaggtcac gggtggacctaaggttccattccaccctggaagagaggacaagcctgagccaccaccagagggtcgcttgcctgatcccactaagggttctg accatttgagagatgtgtttggcaaagctatggggcttactgaccaagatacgttgctctatctggggggtcacactattggagctgcacacaagg agcgttctggatttgagggtccctggacctctaatcctcttattttcgacaactcatacttcacggagttgttgagtggtgagaaggaaggtctcctt cagctaccttctgacaaggctcttttgtctgaccctgtattccgccctctcgttgataaatatgcagcggacgaagatgccttctttgctgattacgct gaggctcaccaaaagctttccgagcttgggtttgctgatgccgaattcagcagggccgaccccaagaagaagaggaaggtggaccccaaga agaagaggaaggtggaccccaagaagaagaggaaggtgtga VStag-APEX2-NLS-NLS Homo sapiens SEQ ID NO 17 atggagagaaaaataagcagaatccaccttgtttctgaacccagtataactcattttctacaagtatcttgggagaaaacactggaatctggttttgt tattacacttactgatggtcattcagcatggactgggacagtttctgaatcagagatttcccaagaagctgatgacatggcaatggaaaaaggga aatatgttggtgaactgagaaaagcattgttgtcaggagcaggaccagctgatgtatacacgtttaatttttctaaagagtcttgttatttcttctttga gaaaaacctgaaagatgtctcattcagacttggttccttcaacctagagaaagttgaaaacccagctgaagtcattagagaacttatttgttattgct tggacaccattgcagaaaatcaagccaaaaatgagcacctgcagaaagaaaatgaaaggcttctgagagattggaatgatgttcaaggacgat XRCC4 Homo sapiens ttgaaaaatgtgtgagtgctaaggaagctttggagactgatctttataagcggtttattctggtgttgaatgagaagaaaacaaaaatcagaagttt gcataataaattattaaatgcagctcaagaacgagaaaaggacatcaaacaagaaggggaaactgcaatctgttctgaaatgactgctgaccga gatccagtctatgatgagagtactgatgaggaaagtgaaaaccaaactgatctctctgggttggcttcagctgctgtaagtaaagatgattccatt atttcaagtcttgatgtcactgatattgcaccaagtagaaaaaggagacagcgaatgcaaagaaatcttgggacagaacctaaaatggctcctca ggagaatcagcttcaagaaaaggaaaagcctgattcttcactacctgagacgtctaaaaaggagcacatctcagctgaaaacatgtctttagaa actctgagaaacagcagcccagaagacctctttgatgagatttaa SEQ ID NO 18 atggacgcacaaacacgacgacgtgagcgtcgcgctgagaaacaagctcaatggaaagctgcaaacggtggatctcctccacatatggctta cccatacgatgttccagattacgctcctccatctcgagctcaagcttcgaattctgcagtcgacggtaccgcgggcatgggagtccccaagtttta cagatggatctcagagcggtatccctgtctcagcgaagtggtgaaagagcatcagattcctgaatttgacaacttgtacctggatatgaatggaat tatacatcagtgctcccatcctaatgatgatgatgttcactttagaatttcagatgataaaatctttactgatatttttcactacctggaggtgttgtttcg cattattaaacccaggaaagtgttctttatggctgtagatggtgtggctcctcgagcaaaaatgaaccagcagcgtgggaggcgttttaggtcag caaaggaggcagaagacaaaattaaaaaggcaatagagaagggagaaactcttcctacagaggccagatttgattccaactgtatcacacca ggaactgaatttatggccaggttacatgaacatctgaagtattttgtaaatatgaaaatttccacagacaagtcatggcaaggagttaccatctactt ctcaggccatgagactcctggagaaggagagcataaaatcatggaatttatcagatccgagaaagcaaagccagatcatgatccaaacacca gacactgtctttatggtttagatgctgacttgattatgcttggattaacaagtcatgaggcacatttttctctcttaagagaagaagttcgatttggtgg caaaaaaacacaacgggtatgtgctccagaagaaactacatttcaccttctacacttgtctttaatgagagagtatattgactatgagttttcagtatt aaaagaaaagatcacatttaaatatgatattgaaaggataatagatgattggattttgatggggtttcttgttggtaatgattttatccctcatctacctc atttacatattaatcatgatgcactgcctcttctttatggaacatatgttaccatcctgccagaacttgggggttatattaatgaaagtgggcacctcaa cttacctcgatttgagaaataccttgtgaaactatcagattttgatcgggagcacttcagtgaagtttttgtggacctaaaatggtttgaaagcaaagt tggtaacaagtacctcaatgaagcagcaggtgtcgcagcagaagaagccaggaactacaaggaaaagaaaaagttaaagggccaggaaaa ttctctgtgttggactgctttagacaaaaatgaaggcgaaatgataacttctaaggataatttagaagatgagactgaagatgatgacctatttgaaa ctgagtttagacaatataaaagaacatattacatgacgaagatgggggttgacgtagtatctgatgactttctggctgatcaagctgcatgttatgtt caggcaatacagtggattttgcactattactatcatggagttcagtcctggagctggtattatccttatcattatgcgcctttcctgtctgatatacaca acatcagtacactcaaaatccattttgaactaggaaaaccttttaagccatttgaacagcttcttgctgtacttccagcagccagcaaaaatttacttc ctgcatgctaccagcatttgatgaccaatgaagactcaccaattatagaatattacccacctgattttaaaactgacctaaatgggaaacaacagg aatgggaagctgtggtgttaatcccttttattgatgagaagcgattattggaagccatggagacatgtaaccactccctcaaaaaggaagagagg aaaagaaaccaacatagtgagtgcctaatgtgctggtatgatagagacacagagtttatctatccttctccatggccagaaaagttccctgccata gaacgatgttgtacaaggtataaaataatatccttagatgcttggcgtgtagacataaacaaaaacaaaataaccagaattgaccagaaagcatt atatttctgtggatttcctactctgaaacacatcagacacaaattttttttgaagaaaagtggtgttcaagtattccagcaaagcagtcgtggagaaa acatgatgttggaaatcttagtggatgcagaatcagatgaacttaccgtagaaaatgtagcttcatcagtgcttggaaaatctgtctttgttaattggc ctcaccttgaggaagctagagtcgtggctgtatcagatggagaaactaagttttacttggaagaacctccaggaacacagaagctttattcagga agaactgccccaccatctaaagtggttcatcttggagataaagaacaatctaactgggcaaaagaagtacaaggaatttcagaacactacctga gaagaaaaggaataataataaatgaaacatctgcagttgtgtatgctcagttactcacaggtcgtaaatatcaaataaatcaaaatggtgaagttc gtctagagaaacagtggtcaaaacaagttgttccttttgtttatcaaactattgtcaaggacatccgagctttcgactcccgtttctccaatatcaaaa cattggatgatttgtttcctctgagaagtatggtctttatgctgggaactccctattatggctgcactggagaagttcaggattcaggtgatgtgatta cagaaggtaggattcgtgtgattttcagcattccatgtgaacccaatcttgatgctttaatacagaaccagcataaatattctataaagtacaaccca ggatatgtgttggccagtcgccttggagtgagtggataccttgtttcaaggtttacaggaagtatttttattggaagaggatctaggagaaaccctc atggagaccataaagcaaatgtgggtttaaatctcaaattcaacaagaaaaatgaggaggtacctggatatactaagaaagttggaagtgaatg gatgtattcatctgcagcagaacaacttctggcagagtacttagagagagctccagaactatttagttatatagccaaaaatagccaagaggatgt gttctatgaagatgacatttggcctggagaaaatgagaatggtgctgaaaaagttcaagaaattattacttggctaaaaggacatcctgtcagtact ttatctcgttcttcttgtgatttacaaattctggatgcagctattgttgagaaaattgaggaagaagtcgaaaagtgcaagcaaagaaagaataataa gaaggtgcgagtaacagtgaaaccccatttgctatacagacctttagaacagcaacatggagtcattcctgatcgggatgcagaattttgtcttttt gaccgtgttgtaaatgtgagagaaaacttctcagttccagttggccttcgaggcaccatcataggaataaaaggagctaatagagaagccgatg tactatttgaagtattatttgatgaagaatttcctggagggttaacaataagatgctcacctggtagaggttatcgactgccaacaagtgccttggtg aacctttctcatgggagtcgctctgaaactggaaatcagaagttgacagccatcgtaaaaccacaaccagctgtacatcaacatagctcaagttc atcagtttcctctgggcatttgggagccctcaaccattcccctcaatcactttttgttcctactcaagtacctactaaagatgatgatgaattctgcaac atttggcagtccttacagggatctggaaagatgcaatactttcagccaactatacaagagaagggtgcagttctacctcaagaaataagccaagt aaatcaacatcataaatctggctttaatgacaacagtgttaaatatcagcaaagaaaacatgaccctcacagaaaatttaaagaagagtgtaaga gtcctaaagctgagtgttggtcccaaaaaatgtccaataagcagcctaactctggaattgagaactttttagcatctttgaatatctccaaagaaaat gaagtacagtcatctcatcatggggagcctccaagtgaagagcatttgtcaccacagtcatttgccatgggaacacggatgcttaaagaaattct aaaaattgatggctctaacactgtggaccataagaatgaaatcaaacagattgctaatgaaatccctgtttcctctaacagaagagatgaatatgg attaccctctcagcctaaacaaaataagaaattagcatcttatatgaacaagcctcacagtgctaatgagtaccataatgttcagtctatggacaata tgtgttggcctgcccccagccagatccctcctgtatccacaccagtaactgaactttctcgaatttgttcccttgttggaatgccacaacctgatttct cctttcttaggatgccacagacaatgaccgtttgccaagtaaaattatctaatggcttactggtacatgggccacagtgccactctgaaaatgaag ccaaagagaaagctgcactttttgctttacaacagttgggctccttaggcatgaatttccctttgccttcacaagtatttgcaaattatccttcagctgt accacctggaaccattcctccagcctttcccccacctactggctgggatcactatggaagcaactatgcattgggggcagctaatataatgccttc V5tag-XRN1 Homo sapiens gtcgtctcatctctttggctcaatgccatggggaccatcggtgccagttcctgggaagcccttccatcatactttatattctgggaccatgcccatg gctgggggaataccagggggtgtgcacaatcagtttatacctctgcaggttactaaaaaaagggttgcaaacaaaaagaactttgagaataagg aagcccagagttctcaagccactccagttcagactagccagccagattcttccaacattgtcaaagtaagtccacgggagagctcatcagcttct ttgaagtcctctccgattgctcaacctgcatcttcttttcaagttgaaactgcctctcaaggccatagtatatctcaccataagtcaacaccaatctctt cttcaagaagaaaatcaagaaaactggctgttaattttggtgtttctaaaccttctgagtaa SEQ ID NO 19 atggagcagctgaacgaactggagctgctgatggagaagagtttttgggaggaggcggagctgccggcggagctatttcagaagaaagtgg tagcttcctttccaagaacagttctgagcacaggaatggataaccggtacctggtgttggcagtcaatactgtacagaacaaagagggaaactgt gaaaagcgcctggtcatcactgcttcacagtcactagaaaataaagaactatgcatccttaggaatgactggtgttctgttccagtagagccagg agatatcattcatttggagggagactgcacatctgacacttggataatagataaagattttggatatttgattctgtatccagacatgctgatttctgg caccagcatagccagtagtattcgatgtatgagaagagctgtcctgagtgaaacttttaggagctctgatccagccacacgccaaatgctaattg gtacggttctccatgaggtgtttcaaaaagccataaataatagctttgccccagaaaagctacaagaacttgcttttcaaacaattcaagaaataag acatttgaaggaaatgtaccgcttaaatctaagtcaagatgaaataaaacaagaagtagaggactatcttccttcgttttgtaaatgggcaggaga tttcatgcataaaaacacttcgactgacttccctcagatgcagctctctctgccaagtgataatagtaaggataattcaacatgtaacattgaagtcg tgaaaccaatggatattgaagaaagcatttggtcccctaggtttggattgaaaggcaaaatagatgttacagttggtgtgaaaatacatcgagggt ataaaacaaaatacaagataatgccgctggaacttaaaactggcaaagaatcaaattctattgaacaccgtagtcaggttgttctgtacactctact aagccaagagagaagagctgatccagaggctggcttgcttctctacctcaagactggtcagatgtaccctgtgcctgccaaccatctagataaa agagaattattaaagctaagaaaccagatggcattctcattgtttcaccgtattagcaaatctgctactagacagaagacacagcttgcttctttgcc acaaataattgaggaagagaaaacttgtaaatattgttcacaaattggcaattgtgctctttatagcagagcagttgaacaacagatggattgtagtt cagtcccaattgtgatgctgcccaaaatagaagaagaaacccagcatctgaagcaaacacacttagaatatttcagcctttggtgtctaatgttaa ccctggagtcacaatcgaaggataataaaaagaatcaccaaaatatctggctaatgcctgcttcggaaatggagaagagtggcagttgcattgg aaacctgattagaatggaacatgtaaagatagtttgtgatgggcaatatttacataatttccaatgtaaacatggtgccatacctgtcacaaatctaat ggcaggtgacagagttattgtaagtggagaagaaaggtcactgtttgctttgtctagaggatatgtgaaggagattaacatgacaacagtaactt gtttattagacagaaacttgtcggtccttccagaatcaactttgttcagattagaccaagaagaaaaaaattgtgatatagataccccattaggaaat ctttccaaattgatggaaaacacgtttgtcagcaaaaaacttcgagatttaattattgactttcgtgaacctcagtttatatcctaccttagttctgttctt ccacatgatgcaaaggatacagttgcctgcattctaaagggtttgaataagcctcagaggcaagcgatgaaaaaggtacttctttcaaaagacta cacactcatcgtgggtatgcctgggacaggaaaaacaactacgatatgtactctcgtaagaattctctacgcctgtggttttagcgttttgttgacca gctatacacactctgctgttgacaatattcttttgaagttagccaagtttaaaataggatttttgcgtttgggtcagattcagaaggttcatccagctat ccagcaatttacagagcaagaaatttgcagatcaaagtccattaaatccttagctcttctagaagaactctacaatagtcaacttatagttgcaacaa catgtatgggaataaaccatccaatattttcccgtaaaatttttgatttttgtattgtggatgaagcctctcaaattagccaaccaatttgtctgggccc cctttttttttcacggagatttgtgttagtgggggaccatcagcagcttcctcccctggtgctaaaccgtgaagcaagagctcttggcatgagtgaa agcttattcaagaggctggagcagaataagagtgctgttgtacagttaaccgtgcagtacagaatgaacagtaaaattatgtccttaagtaataag ctgacctatgagggcaagctggagtgtggatcagacaaagtggccaatgcagtgataaacctacgtcactttaaagatgtgaagctggaactg gaattttatgctgactattctgataatccttggttgatgggagtatttgaacccaacaatcctgtttgtttccttaatacagacaaggttccagcgccag aacaagttgaaaaaggtggtgtgagcaatgtaacagaagccaaactcatagttttcctaacctccatttttgttaaggctggatgcagtccctctga tattggtattattgcaccgtacaggcagcaattaaagatcatcaatgatttattggcacgttctattgggatggtcgaagttaatacagtagacaaat accaaggaagggacaaaagtattgtcctagtatcttttgttagaagtaataaggatggaactgttggtgaactcttgaaagattggcgacgtcttaa tgttgctataaccagagccaaacataaactgattcttctggggtgtgtgccctcactaaattgctatcctcctttggagaagctgcttaatcatttaaa ctcagaaaaattaatcattgatcttccatcaagagaacatgaaagtctttgccacatattgggtgactttcaaagagaataa DNA2 Homo sapiens SEQ ID NO 20 atggaacaaaagttgatttctgaagaagatttgttaagaaagagagggatcctgaatcttctgcgtcggagtgggaaacggcggcgttcagaat caggctcagattcgttctcgggaagcggcggtgacagcagtgccagcccccagttcctctccgggtccgtgctgagcccgccgcccggcctt ggtcgctgcctgaaggccgcagctgcaggagaatgcaagcctacagttcctgactacgaaatagacaagctactattggcaaactggggactt cctaaagcagttctggaaaaataccacagttttggtgtaaaaaagatgtttgaatggcaggcagagtgccttttgcttggacaagtcctggaagg aaagaatttagtttattcagctcctacaagtgctgggaagactcttgtggcagaattacttattttgaagcgggttttggaaatgcggaagaaagctt tgtttattcttccctttgtttctgtggctaaagagaagaaatactacctccagagtctgtttcaggaagtaggaataaaagtagacggttatatgggca gcacctctccatcaaggcatttctcttcattggatattgcagtctgcacaattgagagagccaatggtctgatcaatcgcctcatagaggaaaataa gatggatctgttaggaatggtggttgtggatgaattacatatgctgggagactctcaccgagggtatctgctggaacttttgctgaccaagatttgc tatattactcggaaatcagcatcttgtcaggcagatctagccagttctctgtctaatgctgtgcaaatcgttggcatgagtgctacccttcctaatttg gagcttgtggcttcctggttgaatgctgaactctaccataccgactttcgccctgtaccgcttttggagtcagtaaaagttggaaattccatatatga ctcttcaatgaaacttgtgagggaatttgagcccatgcttcaagtgaagggagatgaggaccatgttgttagcttatgttatgagacgatttgtgata accattcagtattacttttttgtccatcaaagaaatggtgtgagaagctggcagatatcattgctcgagagttttataatctacatcatcaagctgagg gattggtgaaaccctctgaatgcccaccagtaattctggaacaaaaagaactcctggaagtgatggatcagttaagacggttgccttcaggactg gactctgtattacagaaaactgtaccatggggagtagcatttcatcatgcaggtcttacttttgaggagagggatatcattgaaggagcctttcgtc aaggtctcattcgagtcttggcggcaacttctactctttcttctggggtgaatttacctgcacgtcgtgtgattattcgaacccctatttttggtggtcg acctctagatattcttacttataagcagatggttggtcgtgctggcaggaaaggagtggacacagtaggcgagagtatcttaatttgtaagaactct gagaaatcaaaaggcatagctctccttcagggttctctaaagcctgttcgcagctgtctgcaaagacgagaaggagaagaagtaactggcagc atgatacgagctattctggagataatagttggtggagtggcaagtacatcacaagatatgcatacttatgctgcctgcacatttttggctgcaagtat Myc-POLQ-Flag Homo sapiens gaaagaagggaagcaaggaattcagagaaatcaagagtctgttcagcttggagcgattgaggcctgtgtgatgtggctactagaaaatgaattc atccagagtacagaagccagtgatggaacagaaggaaaggtgtatcatccaacacatcttggttcggccactctttcttcttcactttctccagctg atactttagatatttttgctgacctgcaaagagcaatgaagggctttgttttagagaatgatcttcatattctctatctggttacacctatgtttgaggatt ggactactattgattggtatcgatttttctgtttatgggagaagttgccaacttcaatgaaaagggtggcagagctagtgggagttgaagaggggtt cttggcccgttgtgtgaaaggaaaagtagtagccagaactgagagacagcatcgacaaatggccatccataaaaggtttttcaccagtcttgtgc tattagatttaatcagtgaagttcccttaagggaaataaatcagaaatatggatgcaatcgtgggcagattcaatctttgcaacagtcagctgctgtt tatgcagggatgattacagtattttccaaccgtctgggctggcacaacatggaactactactttcccaatttcagaagcgtcttacgtttggcatcca gagggagctgtgtgacctggttcgggtatccttactaaatgctcagagagccagggttctctatgcttctggctttcatactgtggcagaccttgct agagcaaatattgtggaggtggaggtgattctgaaaaatgctgtgcctttcaaaagtgcccggaaggcagtggatgaggaagaggaagcagtt gaagaacgtcgcaatatgcgaactatctgggtgactggcagaaaaggtttaactgaaagggaagcagcagcccttatagtggaagaagccag aatgattctgcagcaggacttagttgaaatgggagtgcaatggaatccatgtgccctgttacattctagtacatgctcattgactcatagtgagtcc gaagtaaaggaacacacatttatatcccaaactaagagttcttataaaaaattaacatcaaagaacaaaagtaacacaatatttagtgattcttatatt aagcattcaccaaatatagtgcaagacttaaataaaagtagagagcatacaagttcctttaattgtaatttccagaatgggaatcaagaacatcag agatgttccattttcagagcaagaaaacgggcctctttagatataaataaagagaagccaggagcctctcagaatgaggggaaaacaagtgata agaaagttgttcagactttttcacagaaaacaaaaaaggcacctttgaatttcaattcagaaaagatgagcagaagttttcgatcttggaaacgtag aaagcatctaaagcgatctagggacagcagccccctgaaagactctggagcgtgtagaatccatttacaaggacagactctgtctaatcctagt ctttgtgaagacccgtttaccttagatgagaagaaaacggaatttagaaattcagggccatttgctaaaaatgtatctttgagtggtaaggaaaaag ataataaaacatcattcccattacaaataaagcaaaattgttcatggaacataacactaactaatgataattttgtggagcatattgtcacaggatctc agagtaaaaatgtgacttgtcaggccactagtgtggttagtgaaaagggcagaggagtagctgttgaggcagaaaaaataaatgaagtgctga tacaaaatggttcaaaaaaccagaatgtttatatgaaacaccatgacatccatccaattaaccagtacctgcgaaagcaatctcatgaacagaca agcactattaccaaacagaaaaatataatagagagacaaatgccctgtgaagcagtcagtagttacataaatagagactcaaatgttactatcaat tgtgaaaggataaagcttaatacagaggaaaataaaccaagtcattttcaggcattaggagatgatataagcagaactgtgatacccagtgaagt acttccatcagctggagcatttagcaaatcagaaggccagcatgagaattttctaaatatttctagactacaagaaaaaacaggtacttatacaaca aacaaaactaaaaataatcatgtttctgacttaggtttagtcctctgtgattttgaagatagtttctatctggatactcagtcagagaaaataatacaac agatggcaactgaaaatgccaaactaggagcaaaggacaccaacctggcagcagggataatgcagaagagcttagtccaacagaactcaat gaactcttttcagaaggagtgtcacattccttttcctgctgaacagcaccctctaggagcgactaagatagatcatttggaccttaagactgtaggt actatgaaacaaagcagtgattcacatggggttgatatcctgactccagaaagcccgattttccattctccaatactattggaggaaaatggtctttt tttaaaaaagaatgaagtttctgttactgattcacaattaaatagttttcttcaaggttatcaaacacaagaaactgtgaaaccagttatacttctgattc ctcaaaagagaactcccactggtgtagaaggagaatgtcttccagttcctgaaacaagtttgaatatgagtgatagtttactatttgatagcttcagt gatgactatctagtaaaagaacaattacctgatatgcaaatgaaagaaccccttccttcagaagtaacatcaaaccattttagtgattctctgtgtcta caagaagacctaattaaaaaatcaaatgtaaatgagaatcaagatacccaccagcagttgacttgttccaatgatgaatctattatattttcagaaat ggattctgttcagatggttgaagctttggacaatgtggatatatttcctgtccaagagaagaatcatactgtagtatctcctagagcattagaactaa gtgatccagtacttgatgagcaccaccaaggtgatcaagatggaggagatcaagatgaaagggctgaaaaatcaaaattaactgggaccagg caaaatcattcattcatttggtcaggggcatcatttgatctaagtccaggactgcaaaggattttagataaagtatccagtcctctagaaaatgaaaa gctaaaatcaatgactataaacttttccagtttgaatagaaaaaatacagagttaaatgaagaacaagaagttatttcaaacttggagacaaaacaa gtgcagggaatttcattttcttctaataatgaagtaaaaagcaagattgagatgctagaaaacaatgccaatcatgatgaaacctcatccctcttacc tcgtaaagaaagtaatatagttgatgataatggtctcattcctcctacacccattccaacatctgcttctaagctgacatttccagggattcttgaaac acctgtaaacccttggaaaactaataatgttttacaacctggtgaaagttatttatttggctcaccttcagatattaaaaaccacgatttaagtccagg gagtagaaatgggttcaaagacaacagccctattagtgacacaagcttttcacttcagttatcacaggatggattacagttaactccagcctcaag cagttcagaaagtttgtccataattgatgtagcaagtgaccaaaatcttttccaaacattcattaaggagtggcggtgcaaaaagcgattttccatct cactggcttgtgaaaagattagaagtttgacatcttctaaaactgctactattggcagtaggtttaagcaagctagctcacctcaggaaattcctatt agagatgatggatttcccattaaaggttgtgatgacaccttggtggttggactggcagtatgctggggtggaagggatgcctattatttttcactgc agaaggaacaaaagcattctgaaattagtgccagtttggttccaccttctttagatccaagcctgactttgaaagacaggatgtggtaccttcaatc ttgcttgcgaaaggaatctgataaagaatgttctgttgtcatctatgacttcatccagagctataaaattcttcttctttcttgtggcatctccttggagc aaagttatgaagatcctaaggtggcatgctggttactagatccagattctcaggagccgactcttcatagcatagttaccagttttcttcctcatgag cttccactcctagaagggatggagaccagccaagggattcaaagcctggggctaaatgctggcagtgagcattctgggcgatacagagcatct gtggagtccattctcatcttcaactctatgaatcagctcaactctttgttgcagaaggaaaaccttcaagatgttttccgtaaggtggaaatgccctct cagtactgcttggccttgctagaactaaatggaattggctttagtactgcagaatgtgaaagtcagaaacatataatgcaagccaagctggatgc aattgagacccaggcctatcaactagctggccacagtttttctttcaccagttcagatgacatcgctgaggttttatttttggaattgaagttgccccc aaatagagagatgaaaaaccaaggcagcaagaaaactctgggttctaccagaagagggattgacaatggacgcaagctaaggctgggaag acagttcagcactagtaaggacgttttaaataaattaaaggcattacatcctttaccaggcttgatattagaatggagaagaatcactaatgctatta ccaaagtggtctttccccttcagcgggaaaagtgtcttaatccttttcttggaatggaaagaatctatcctgtatcacagtcgcacactgctacagg acgaataacctttacagaaccaaatattcagaatgtgccaagagattttgaaatcaaaatgccaacactagtaggagaaagcccaccttctcaag ctgtaggcaaaggcctacttcccatgggcagaggaaaatataagaagggtttcagcgtgaatcctagatgccaggcacagatggaggagaga gctgcagacagaggaatgccattttcaattagcatgcgacatgcctttgtgcctttcccaggtggttcaatactggctgctgactactctcagcttg aactgaggatcttggctcatttatcccatgatcgtcgtctcattcaagtgttaaacactggagctgatgttttcaggagcattgcagcagagtggaa gatgattgagccagagtctgttggggatgatctgaggcagcaggcaaaacagatttgctatgggatcatttatggaatgggagctaaatctttgg gagagcagatgggcattaaagaaaatgatgctgcatgctatattgactccttcaaatccagatacacagggattaatcaattcatgacagagaca gtgaagaattgtaaaagagacggatttgttcagaccattttgggaaggcgtagatatttgccaggaatcaaagacaacaacccttatcgtaaagc tcacgctgagcgtcaagctatcaacacaatagtccaaggatcagcagctgatattgtcaaaatagccacagttaacattcagaagcaattagaga ccttccactcaaccttcaaatcccatggtcatcgagagggtatgctccaaagtgaccgaacaggattgtcacgaaagagaaaactgcaaggga tgttctgcccaatcagaggaggcttcttcatccttcaactccatgatgaactcctatatgaagtggcagaagaagatgttgttcaggtagctcagatt gtcaagaatgaaatggaaagtgctgtaaaactgtctgtgaaattgaaagtgaaagtgaaaataggcgccagctggggagagctaaaggacttt gatgtgcccgggatggactacaaagacgatgacgacaagtaa SEQ ID NO 21 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgctagcaaacggaaggcgccgcaggagactctcaacgggggaatcaccgacatgctcacag aactcgcaaactttgagaagaacgtgagccaagctatccacaagtacaatgcttacagaaaagcagcatctgttatagcaaaatacccacacaa aataaagagtggagctgaagctaagaaattgcctggagtaggaacaaaaattgctgaaaagattgatgagtttttagcaactggaaaattacgta aactggaaaagattcggcaggatgatacgagttcatccatcaatttcctgactcgagttagtggcattggtccatctgctgcaaggaagtttgtag atgaaggaattaaaacactagaagatctcagaaaaaatgaagataaattgaaccatcatcagcgaattgggctgaaatattttggggactttgaa aaaagaattcctcgtgaagagatgttacaaatgcaagatattgtactaaatgaagttaaaaaagtggattctgaatacattgctacagtctgtggca gtttcagaagaggtgcagagtccagtggtgacatggatgttctcctgacccatcccagcttcacttcagaatcaaccaaacagccaaaactgtta catcaggttgtggagcagttacaaaaggttcattttatcacagataccctgtcaaagggtgagacaaagttcatgggtgtttgccagcttcccagt aaaaatgatgaaaaagaatatccacacagaagaattgatatcaggttgatacccaaagatcagtattactgtggtgttctctatttcactgggagtg atattttcaataagaatatgagggctcatgccctagaaaagggtttcacaatcaatgagtacaccatccgtcccttgggagtcactggagttgcag gagaacccctgccagtggatagtgaaaaagacatctttgattacatccagtggaaataccgggaacccaaggaccggagcgaatga POLB Homo sapiens SEQ ID NO 22 atggctactggacaggatcgagtggttgctctcgtggacatggactgtttttttgttcaagtggagcagcggcaaaatcctcatttgaggaataaa ccttgtgcagttgtacagtacaaatcatggaagggtggtggaataattgcagtgagttatgaagctcgtgcatttggagtcactagaagtatgtgg gcagatgatgctaagaagttatgtccagatcttctactggcacaagttcgtgagtcccgtgggaaagctaacctcaccaagtaccgggaagcca gtgttgaagtgatggagataatgtctcgttttgctgtgattgaacgtgccagcattgatgaggcttacgtagatctgaccagtgctgtacaagaga gactacaaaagctacaaggtcagcctatctcggcagacttgttgccaagcacttacattgaagggttgccccaaggccctacaacggcagaag agactgttcagaaagaggggatgcgaaaacaaggcttatttcaatggctcgattctcttcagattgataacctcacctctccagacctgcagctca ccgtgggagcagtgattgtggaggaaatgagagcagccatagagagggagactggttttcagtgttcagctggaatttcacacaataaggtcct ggcaaaactggcctgtggactaaacaagcccaaccgccaaaccctggtttcacatgggtcagtcccacagctcttcagccaaatgcccattcg caaaatccgtagtcttggaggaaagctaggggcctctgtcattgagatcctagggatagaatacatgggtgaactgacccagttcactgaatcc cagctccagagtcattttggggagaagaatgggtcttggctatatgccatgtgccgagggattgaacatgatccagttaaacccaggcaactac ccaaaaccattggctgtagtaagaacttcccaggaaaaacagctcttgctactcgggaacaggtacaatggtggctgttgcaattagcccagga actagaggagagactgactaaagaccgaaatgataatgacagggtagccacccagctggttgtgagcattcgtgtacaaggagacaaacgcc tcagcagcctgcgccgctgctgtgcccttacccgctatgatgctcacaagatgagccatgatgcatttactgtcatcaagaactgtaatacttctg gaatccagacagaatggtctcctcctctcacaatgcttttcctctgtgctacaaaattttctgcctctgccccttcatcttctacagacatcaccagctt cttgagcagtgacccaagttctctgccaaaggtgccagttaccagctcagaagctaagacccagggaagtggcccagcggtgacagccacta agaaagcaaccacgtctctggaatcattcttccaaaaagctgcagaaaggcagaaagttaaagaagcttcgctttcatctcttactgctcccactc aggctcccatgagcaattcaccatccaagccctcattaccttttcaaaccagtcaaagtacaggaactgagcccttctttaagcagaaaagtctgc ttctaaagcagaaacagcttaataattcttcagtttcttccccccaacaaaacccatggtccaactgtaaagcattaccaaactctttaccaacagag tatccagggtgtgtccctgtttgtgaaggggtgtcgaagctagaagaatcctctaaagcaactcctgcagagatggatttggcccacaacagcc aaagcatgcacgcctcttcagcttccaaatctgtgctggaggtgactcagaaagcaaccccaaatccaagtcttctagctgctgaggaccaagt gccctgtgagaagtgtggctccctggtaccggtatgggatatgccagaacacatggactatcattttgcattggagttgcagaaatcctttttgca gccccactcttcaaacccccaggttgtttctgccgtatctcatcaaggcaaaagaaatcccaagagccctttggcctgcactaataaacgcccca ggcctgagggcatgcaaacattggaatcattttttaagccattaacacattag POLH Homo sapiens SEQ ID NO 23 atggctagccgcctgctctggaggaaggtggccggcgccaccgtcgggccagggccggttccagctccggggcgctgggtctccagctcc gtccccgcgtccgaccccagcgacgggcagcggcggcggcagcagcagcagcagcagcagcagcagcagcaacagcagcctcagcag ccgcaagtgctatcctcggagggcgggcagctgcggcacaacccattggacatccagatgctctcgagagggctgcacgagcaaatcttcg ggcaaggaggggagatgcctggcgaggccgcggtgcgccgcagcgtcgagcacctgcagaagcacgggctctgggggcagccagccg tgcccttgcccgacgtggagctgcgcctgccgcccctctacggggacaacctggaccagcacttccgcctcctggcccagaagcagagcct gccctacctggaggcggccaacttgctgttgcaggcccagctgcccccgaagcccccggcttgggcctgggcggagggctggacccggta cggccccgagggggaggccgtacccgtggccatccccgaggagcgggccctggtgttcgacgtggaggtctgcttggcagagggaacttg ccccacattggcggtggccatatccccctcggcctggtattcctggtgcagccagcggctggtggaagagcgttactcttggaccagccagct gtcgccggctgacctcatccccctggaggtccctactggtgccagcagccccacccagagagactggcaggagcagttagtggtggggcac aatgtttcctttgaccgagctcatatcagggagcagtacctgatccagggttcccgcatgcgtttcctggacaccatgagcatgcacatggccat ctcagggctaagcagcttccagcgcagtctgtggatagcagccaagcagggcaaacacaaggtccagccccccacaaagcaaggccagaa gtcccagaggaaagccagaagaggcccagcgatctcatcctgggactggctggacatcagcagtgtcaacagtctggcagaggtgcacag actttatgtaggggggcctcccttagagaaggagcctcgagaactgtttgtgaagggcaccatgaaggacattcgtgagaacttccaggacctg POLG Homo sapiens atgcagtactgtgcccaggacgtgtgggccacccatgaggttttccagcagcagctaccgctcttcttggagaggtgtccccacccagtgactc tggccggcatgctggagatgggtgtctcctacctgcctgtcaaccagaactgggagcgttacctggcagaggcacagggcacttatgaggag ctccagcgggagatgaagaagtcgttgatggatctggccaatgatgcctgccagctgctctcaggagagaggtacaaagaagacccctggct ctgggacctggagtgggacctgcaagaatttaagcagaagaaagctaagaaggtgaagaaggaaccagccacagccagcaagttgcccat cgagggggctggggcccctggtgatcccatggatcaggaagacctcggcccctgcagtgaggaggaggagtttcaacaagatgtcatggcc cgcgcctgcttgcagaagctgaaggggaccacagagctcctgcccaagcggccccagcaccttcctggacaccctggatggtaccggaag ctctgcccccggctagacgaccctgcatggaccccgggccccagcctcctcagcctgcagatgcgggtcacacctaaactcatggcacttac ctgggatggcttccctctgcactactcagagcgtcatggctggggctacttggtgcctgggcggcgggacaacctggccaagctgccgacag gtaccaccctggagtcagctggggtggtctgcccctacagagccatcgagtccctgtacaggaagcactgtctcgaacaggggaagcagca gctgatgccccaggaggccggcctggcggaggagttcctgctcactgacaatagtgccatatggcaaacggtagaagaactggattacttag aagtggaggctgaggccaagatggagaacttgcgagctgcagtgccaggtcaacccctagctctgactgcccgtggtggccccaaggacac ccagcccagctatcaccatggcaatggaccttacaacgacgtggacatccctggctgctggtttttcaagctgcctcacaaggatggtaatagct gtaatgtgggaagcccctttgccaaggacttcctgcccaagatggaggatggcaccctgcaggctggcccaggaggtgccagtgggccccg tgctctggaaatcaacaaaatgatttctttctggaggaacgcccataaacgtatcagctcccagatggtggtgtggctgcccaggtcagctctgc cccgtgctgtgatcaggcaccccgactatgatgaggaaggcctctatggggccatcctgccccaagtggtgactgccggcaccatcactcgc cgggctgtggagcccacatggctcaccgccagcaatgcccggcctgaccgagtaggcagtgagttgaaagccatggtgcaggccccacct ggctacacccttgtgggtgctgatgtggactcccaagagctgtggattgcagctgtgcttggagacgcccactttgccggcatgcatggctgca cagcctttgggtggatgacactgcagggcaggaagagcaggggcactgatctacacagtaagacagccactactgtgggcatcagccgtga gcatgccaaaatcttcaactacggccgcatctatggtgctgggcagccctttgctgagcgcttactaatgcagtttaaccaccggctcacacagc aggaggcagctgagaaggcccagcagatgtacgctgccaccaagggcctccgctggtatcggctgtcggatgagggcgagtggctggtga gggagttgaacctcccagtggacaggactgagggtggctggatttccctgcaggatctgcgcaaggtccagagagaaactgcaaggaagtc acagtggaagaagtgggaggtggttgctgaacgggcatggaaggggggcacagagtcagaaatgttcaataagcttgagagcattgctacgt ctgacataccacgtaccccggtgctgggctgctgcatcagccgagccctggagccctcggctgtccaggaagagtttatgaccagccgtgtga attgggtggtacagagctctgctgttgactacttacacctcatgcttgtggccatgaagtggctgtttgaagagtttgccatagatgggcgcttctg catcagcatccatgacgaggttcgctacctggtgcgggaggaggaccgctaccgcgctgccctggccttgcagatcaccaacctcttgacca ggtgcatgtttgcctacaagctgggtctgaatgacttgccccagtcagtcgcctttttcagtgcagtcgatattgaccggtgcctcaggaaggaag tgaccatggattgtaaaaccccttccaacccaactgggatggaaaggagatacgggattccccagggtgaagcgctggatatttaccagataat tgaactcaccaaaggctccttggaaaaacgaagccagcctggaccatag SEQ ID NO 24 atggaaaattatgaggcattggtaggctttgatctctgtaatacaccgctctccagtgttgctcagaagattatgtctgctatgcattcaggtgattta gtggattctaagacttggggaaagagtacagagactatggaagtgataaacaagtccagtgttaagtattcagtacaacttgaagacaggaaga ctcaatcaccagaaaaaaaggatcttaaatctttaagaagtcagacatcaagaggttctgccaagctgtctcctcagtccttcagtgtcaggctca cagatcagctgtctgctgaccaaaaacagaagagcatcagctcattgactctttcaagttgtttaattccacagtataatcaagaggcttcagttcta cagaaaaaggggcataaaagaaagcatttcctaatggagaatataaataatgaaaataaaggaagcattaatcttaaaagaaaacatattacata taataatttgtcagagaaaacaagtaaacaaatggcattggaagaagatactgatgacgccgaaggctacctaaattctgggaactcaggagca ttgaaaaaacatttttgtgatattaggcatttggatgattgggcaaaaagccagctgattgaaatgctcaaacaggcagcagccctggtgataact gtgatgtatactgatggttccacccagctaggagctgaccagacccccgtttcttctgttagaggaattgtggtgttagtaaaacgccaagcaga gggtggccatggctgtccagatgccccggcctgtggtcctgttctggagggctttgtgtcagatgatccatgcatctacattcaaatagagcactc tgctatctgggaccaagaacaggaggcacatcaacaatttgcccggaacgtgctatttcaaacaatgaaatgtaaatgtcctgttatttgttttaatg ctaaggattttgtgagaaxagtgctgcagttttttggcaatgatggcagttggaagcatgttgctgattttatagggctagatcccagaattgctgcat ggcttatagatcctagtgatgccacaccctcttttgaagatttagtagaaaaatactgtgaaaaatccattacagttaaagtgaacagcacatatgg aaattcctcaagaaatattgtgaatcagaatgtacgtgagaacctgaagacactctacagacttacaatggacctttgctctaaactgaaggattat ggtttatggcaactatttcgtactttggagcttcctctgataccaattttggcagtgatggaaagccatgccattcaggtgaacaaagaggagatgg agaagacgtcagcacttcttggggctcgtctcaaggaattggagcaagaagctcattttgttgcaggagaacggtttcttataacgagcaataac cagcttcgagagatcctctttggcaagttaaagctgcacctgctgagtcaaaggaacagtctccccagaacggggttgcagaaatacccgtcta catcagaagcagtgttaaatgctctgcgagaccttcatccattacccaagataattttggaatacaggcaggttcacaagatcaagtcaacctttgt agatggattactagcttgcatgaaaaagggctccatttcctctacatggaatcagactggaactgtgactggaagactttcagccaagcatcctaa tatccaaggtatctccaagcacccaattcagattactacacctaagaattttaaaggtaaagaagacaagattctcacgatctccccgagggccat gtttgtttcatccaaaggccacacctttctagcagcagacttttcacagattgaattgcgcattcttacacatttatctggagatccggaacttctgaa gttattccaggaatctgaaagagatgatgtattttctactctgacttcacagtggaaggatgtgcccgtggaacaggtgacacacgcagacagag agcaaaccaagaaggtggtgtacgcggtggtctatggagcagggaaggagcggctggctgcttgccttggagttcctattcaggaagctgcc cagtttttggagagttttttgcagaagtacaagaaaatcaaggacttcgcccgagcagctattgcccagtgtcaccagacaggctgtgtggtgtc catcatgggcagaaggagacccctgccaaggattcacgctcatgaccagcaactccgggcacaagcagagcgacaggcagtgaacttcgt ggtgcaaggctccgctgctgacctctgcaagctggccatgatccatgtcttcactgcagtggctgcttcccacaccttgacggccaggctggtg gcccagatccatgatgagctgctgtttgaagtggaagatccgcagatcccggagtgtgcagctctcgtcaggaggaccatggagtccttggaa caggtgcaggcattggagctgcagcttcaggtacccctcaaggtgagcctgagtgccggccgctcatggggacacctggtgccactgcagg POLN Homo sapiens aggcctggggccctccgccaggcccatgtcgcactgagtctcccagcaacagcctggctgcccctgggtcccctgccagcacccagccccc acccctgcatttttcgccttcattttgtctgtag SEQ ID NO 25 atggcttccccttgtcctgaagaagcagctatgagaagagaggtggtgaaacggatcgaaactgtggtgaaagacctttggccgacggctgat gtacagatatttggcagctttagtacaggtctttatcttccaactagcgacatagacctggtggtcttcgggaaatgggagcgtcctcctttacagct gctggagcaagccctgcggaagcacaacgtggctgagccgtgttccatcaaagtccttgacaaggctacggtaccaataataaagctcacag atcaggagactgaagtgaaagttgacatcagctttaacatggagacgggcgtccgggcagcggagttcatcaagaattacatgaagaaatattc attgctgccttacttgattttagtattgaaacagttccttctgcagagggacctgaatgaagtttttacaggtggaattagctcatacagcctaatttta atggccattagctttctacagttgcatccaagaattgatgcccggagagctgatgaaaaccttggaatgcttcttgtagaattttttgaactctatggg agaaattttaattacttgaaaaccggtattagaatcaaagaaggaggtgcctatatcgccaaagaggagatcatgaaagccatgaccagcgggt acagaccgtcgatgctgtgcattgaggaccccctgctgccagggaatgacgttggccggagctcctatggcgccatgcaggtgaagcaggtc ttcgattatgcctacatagtgctcagccatgctgtgtcaccgctggccaggtcctatccaaacagagacgccgaaagtactttaggaagaatcat caaagtaactcaggaggtgattgactaccggaggtggatcaaagagaagtggggcagcaaagcccacccgtcgccaggcatggacagcag gatcaagatcaaagagcgaatagccacatgcaatggggagcagacgcagaaccgagagcccgagtctccctatggccagcgcttgactttgt cgctgtccagcccccagctcctgtcttcaggctcctcggcctcttctgtgtcttcactttctgggagtgacgttgattcagacacaccgccctgcac aacgcccagtgtttaccagttcagtctgcaagcgccagctcctctcatggccggcttacccaccgccttgccaatgcccagtggcaaacctcag cccaccacttccagaacactgatcatgacaaccaacaatcagaccaggtttactatacctccaccgaccctaggggttgctcctgttccttgcag acaagctggtgtagaaggaactgcgtctttgaaagccgtccaccacatgtcttccccggccattccctcagcgtcccccaacccgctctcgagc cctcatctgtatcataagcacaacggcatgaaactgtccatgaagggctctcacggccacacccaaggcggcggctacagctctgtgggtagc ggaggtgtgcggccccctgtgggcaacaggggacaccaccagtataaccgcaccggctggaggaggaaaaaacacacacacacacggg acagtctgcccgtgagcctcagcagataa TENT4A Homo sapiens SEQ ID NO 26 atggctgcctcacaaacttcacaaactgttgcatctcacgttccttttgcagatttgtgttcaactttagaacgaatacagaaaagtaaaggacgtgc agaaaaaatcagacacttcagggaatttttagattcttggagaaaatttcatgatgctcttcataagaaccacaaagatgtcacagactctttttatcc agcaatgagactaattcttcctcagctagaaagagagagaatggcctatggaattaaagaaactatgcttgctaagctttatattgagttgcttaattt acctagagatggaaaagatgccctcaaacttttaaactacagaacacccactggaactcatggagatgctggagactttgcaatgattgcatattt tgtgttgaagccaagatgtttacagaaaggaagtttaaccatacagcaagtaaacgaccttttagactcaattgccagcaataattctgctaaaaga aaagacctaataaaaaagagccttcttcaacttataactcagagttcagcacttgagcaaaagtggcttatacggatgatcataaaggatttaaag cttggtgttagtcagcaaactatcttttctgtttttcataatgatgctgctgagttgcataatgtcactacagatctggaaaaagtctgtaggcaactgc atgatccttctgtaggactcagtgatatttctatcactttattttctgcatttaaaccaatgctagctgctattgcagatattgagcacattgagaaggat atgaaacatcagagtttctacatagaaaccaagctagatggtgaacgtatgcaaatgcacaaagatggagatgtatataaatacttctctcgaaat ggatataactacactgatcagtttggtgcttctcctactgaaggttctcttaccccattcattcataatgcattcaaagcagatatacaaatctgtattct tgatggtgagatgatggcctataatcctaatacacaaactttcatgcaaaagggaactaagtttgatattaaaagaatggtagaggattctgatctg caaacttgttattgtgtttttgatgtattgatggttaataataaaaagctagggcatgagactctgagaaagaggtatgagattcttagtagtatttttac accaattccaggtagaatagaaatagtgcagaaaacacaagctcatactaagaatgaagtaattgatgcattgaatgaagcaatagataaaaga gaagagggaattatggtaaaacaacctctatccatctacaagccagacaaaagaggtgaagggtggttaaaaattaaaccagagtatgtcagtg gactaatggatgaattggacattttaattgttggaggatattggggtaaaggatcacggggtggaatgatgtctcattttctgtgtgcagtagcaga gaagccccctcctggtgagaagccatctgtgtttcatactctctctcgtgttgggtctggctgcaccatgaaagaactgtatgatctgggtttgaaat tggccaagtattggaagccttttcatagaaaagctccaccaagcagcattttatgtggaacagagaagccagaagtatacattgaaccttgtaatt ctgtcattgttcagattaaagcagcagagatcgtacccagtgatatgtataaaactggctgcaccttgcgttttccacgaattgaaaagataagag atgacaaggagtggcatgagtgcatgaccctggacgacctagaacaacttagggggaaggcatctggtaagctcgcatctaaacacctttatat aggtggtgatgatgaaccacaagaaaaaaagcggaaagctgccccaaagatgaagaaagttattggaattattgagcacttaaaagcacctaa ccttactaacgttaacaaaatttctaatatatttgaagatgtagagttttgtgttatgagtggaacagatagccagccaaagcctgacctggagaac agaattgcagaatttggtggttatatagtacaaaatccaggcccagacacgtactgtgtaattgcagggtctgagaacatcagagtgaaaaacat aattttgtcaaataaacatgatgttgtcaagcctgcatggcttttagaatgttttaagaccaaaagctttgtaccatggcagcctcgctttatgattcat atgtgcccatcaaccaaagaacattttgcccgtgaatatgattgctatggtgatagttatttcattgatacagacttgaaccaactgaaggaagtatt ctcaggaattaaaaattctaacgagcagactcctgaagaaatggcttctctgattgctgatttagaatatcggtattcctgggattgctctcctctcag tatgtttcgacgccacaccgtttatttggactcgtatgctgttattaatgacctgagtaccaaaaatgaggggacaaggttagctattaaagccttgg agcttcggtttcatggagcaaaagtagtttcttgtttagctgagggagtgtctcatgtaataattggggaagatcatagtcgtgttgcagattttaaag cttttagaagaacttttaagagaaagtttaaaatcctaaaagaaagttgggtaactgattcaatagacaagtgtgaattacaagaagaaaaccagta tttgatttaa DNA Ligase 4 Homo sapiens SEQ ID NO 27 atgggctccgccgcctgcccccggggagccttgccggagctcgcgccctgctgccagcctcgcgagcagtcgcagccccacacgcgatgg gacgcgggctgtgggattcagcaccccgggggcgaggaattcaggaccctcggcggggcaagggcctatagggttccgaactcgcagga gggtcgctcctcccctactcgctttttcccggcaccggaaggccccgcccactgctttgtttcctctccagaccgcgcattttgggtctcggaaga ggttcagaggctgttgttgagcaatgcatgccagccaaaagaatgcaatggtgtaaagattccagttgatgccagtaaacctaatccaaatgatg tggagtttgataatctgtatttggatatgaatggaatcatccatccctgtactcatcctgaagacaaaccagcaccaaaaaatgaagatgaaatgat ggttgcaatttttgagtacattgacagacttttcagtattgtaagaccaagaagacttctctacatggcaatagatggagtggcaccacgtgctaaa atgaaccagcagcgttcaaggaggttcagggcatcaaaagaaggaatggaagcagcagtcgagaagcagcgagtcagggaagaaatattg XRN Homo sapiens gcaaaaggtggctttcttcctccagaagaaataaaagaaagatttgacagcaactgtattacaccaggaactgaattcatggacaatcttgctaaa tgccttcgctattacatagctgatcgtttaaataatgaccctgggtggaaaaatttgacagttattttatctgatgctagtgctcctggtgaaggagaa cataaaatcatggattacattagaaggcaaagagcccagcctaaccatgacccaaatactcatcattgtttatgtggagcagatgctgatctcatta tgcttggccttgccacacatgaaccgaactttaccattattagagaagaattcaaaccaaacaagcccaaaccatgtggtctttgtaatcagtttgg acatgaggtcaaagattgtgaaggtttgccaagagaaaagaagggaaagcatgatgaacttgccgatagtcttccttgtgcagaaggagagttt atcttccttcggcttaatgttcttcgtgagtatttggaaagagaactcacaatggccagcctaccattcacatttgatgttgagaggagcattgatga ctgggttttcatgtgcttctttgtgggaaatgacttcctccctcatttgccatcgttagagattagggaaaatgcaattgaccgtttggttaacatatac aaaaatgtggtacacaaaactgggggttaccttacagaaagtggttatgtcaatctgcaaagagtacagatgatcatgttagcagttggtgaagtt gaggatagcatttttaaaaagagaaaggatgatgaggacagttttagaagacgacagaaagaaaaaagaaagagaatgaagagagatcaac cagctttcactcctagtggaatattaactcctcatgccttgggttcaagaaattcaccaggttctcaagtagccagtaatccgagacaagcagccta tgaaatgaggatgcagaataactctagtccttcgatatctcctaatacgagtttcacatctgatggctccccgtctccattaggaggaattaagcga aaagcagaagacagtgacagtgaacctgagccagaggataatgtcaggttatgggaagctggctggaagcagcggtactacaagaacaaat ttgatgtggatgcagctgatgagaaattccgtcggaaagttgtgcagtcgtacgttgaaggactttgctgggttcttagatattattaccagggctgt gcttcctggaagtggtattatccatttcattatgcaccatttgcttcagactttgaaggcattgcagacatgccatctgattttgagaagggtacgaaa ccgtttaaaccactagaacaacttatgggggtatttccagctgcaagtggtaattttctacctccatcatggcggaagctcatgagtgatcctgattc tagtataattgacttctatcctgaagattttgctattgatttgaatgggaagaaatatgcatggcaaggtgttgctctcttgccattcgtggatgagcg aaggctacgagctgccctagaagaggtatacccagacctcactccagaagagaccagaagaaacagccttggaggtgatgtcttatttgtggg gaaacatcacccactccatgacttcattttagagctgtaccagacaggttccacagagccagtggaggtaccccctgaactatgtcatgggattc aaggaaagttttctttggatgaagaagccattcttccagatcaaatagtatgttctcctgttcctatgttaagggatctgacacagaacactgtagtca gtattaattttaaagacccacagtttgctgaagattacatttttaaagctgtaatgcttccaggagcaagaaagccagcagcagtactgaaacctag tgactgggaaaaatccagcaatggacggcagtggaagcctcagcttggctttaaccgtgaccggaggcctgtgcacctggatcaggcagcct tcaggactttgggccatgtgatgccaagaggctcaggaactggcatttacagcaatgctgcaccaccacctgtgacttaccagggaaacttata caggccgcttttgagaggacaagcccagattccaaaacttatgtcaaatatgaggccccaggattcctggcgaggtcctcctccccttttccagc agcaaaggtttgacagaggcgttggggctgaacctctgctcccatggaaccggatgctgcaaacccagaatgcagccttccagccaaaccag taccagatgctagctgggcctggtgggtatccacccagacgagatgatcgtggagggagacagggatatcccagagaaggaaggaaatacc ctttgccaccaccctcaggaagatacaattggaattaa SEQ ID NO 28 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagcctcattgaagggaaaattttttgcatttctcccgaaccctaacacctcatccaataagttttttaagtcaa ttcttgagaagaaaggagctacaattgtatcatctattcaaaattgcctccagagttctaggaaagaggtcattatccttatagaggacagtttcgtc gacagtgacatgcacctgacacagaaggacatttttcaacgcgaggctggcttgaacgacgtagacgagtttttgggtaaaattgaacaatccg gcatccagtgcgttaagactagctgcattaccaagtgggttcaaaacgacaaattcgcttttcaaaaggacgatttgattaagttccaaccgagtat catagtcattagtgacaatgccgatgatggacagagtagcactgacaaagaaagcgaaatctcaactgacgtagaatcagagcgaaacgatg actcaaacaacaaagacatgattcaggcctccaaaccgctcaaacggttgcttcaggaggataaaggtcgcgcttcccttgttaccgataaaac caagtataaaaataatgaacttataataggcgcgcttaaacgacttaccaagaagtacgagattgagggtgaaaaattccgagctcggtcctacc ggctcgctaaacaatctatggaaaattgtgatttcaatgttagaagcggagaggaagcacatacaaagttgagaaacatcggtcctagtattgct aaaaaaattcaggtcattcttgatacgggagttctcccgggtctcaacgattccgttggccttgaagacaagctgaaatattttaagaactgctatg gaatcgggtcagagatagcaaaacggtggaatctccttaactttgagtcattttgcgtggctgctaagaaagaccccgaggaatttgtgtccgatt ggacgatattgttcgggtggagttattatgatgattggctttgcaaaatgtccaggaatgaatgcttcgcccatcttaagaaggtccaaaaggcttt gcgcggaatcgaccccgaatgtcaggtcgagcttcaagggtcatacaatcggggttactcaaaatgcggggatatagatctcctcttttttaagc cattctgcaacgataccactgaactcgctaagatcatggagacactctgcataaagctttataaagatgggtatatacattgcttcttgcaattgacg cccaacttggagaagctttttcttaaaagaattgttgaacggttccggacagccaagattgttggctatggagaacgaaaacgctggtattcatca gaaattatcaagaaattctttatgggagtgaagttgtccccccgcgagctcgaagaattgaaggagatgaaaaacgacgagggaaccctgttga tcgaggaagaagaagaggaaacgaagctgaagcccattgaccagtacatgagcctgaacgctaaagacggaaactactgccgaaggttgg attttttctgttgtaagtgggacgagctgggggcggggaggatacactatacgggtagcaaagagtataataggtggataaggatactcgccgc gcaaaaagggttcaaactgacccagcatggacttttccggaacaacatactcctggagtctttcaacgaaaggcgaatcttcgaactcctgaac cttaagtatgccgagccggagcaccgcaatatagagtgggaaaaaaagacgggatga 3xFlag_NLS_ PolIV Escherichia coli SEQ ID NO 29 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccctgcctagtcagagccccgcaatatttacagtgagtcgccttaaccaaaccgttcgactgttgctc gagcacgaaatggggcaagtctggatctccggggaaatatcaaattttacgcagccagcctccggtcactggtacttcactcttaaagatgaca cggcgcaagtacgctgcgccatgtttcggaacagcaatagacgagtgacgttccggccacaacatggacagcaagtactcgtcagggccaat atcactctttatgagccgcgcggtgactatcaaataattgtcgaatctatgcaacccgcgggggagggtttgctccagcaaaagtatgagcaact caaagcgaagctccaggcagaaggcctgttcgaccagcagtataaaaaaccgctcccgtcacccgctcattgtgttggcgtcataacctctaa gacgggtgctgcgttgcacgacattcttcatgtgcttaagcgccgagacccatctctgcctgttatcatctacccagcggccgttcaaggcgatg acgctcctgggcagatagtaagagcaatagaactggcgaatcagcggaacgaatgtgatgtgctgatcgttgggcgcggcggagggagctt ggaagatctttggtccttcaacgatgagcgcgtcgcacgggcaatcttcaccagccggataccggtagtttcagcggtggggcatgagacgga cgtcacaatcgccgattttgtagccgacctgagagcaccgacgccatcagcggcagcagaagtcgtcagccgcaatcagcaggagctgctc 3xFlag_NLS_ XseA Escherichia coli aggcaggtccagagcacccggcaacgcctcgagatggcgatggattactatcttgccaatcgaacacgacgattcacccagattcaccaccg gttgcagcagcaacatccccaacttcggctggcccgacagcaaacaatgctggaacgcctccagaaacggatgagttttgctctggaaaatca gttgaagcgaactggtcaacagcagcaaagactgactcagcgcctcaatcagcaaaatccccaacctaagatccatcgggcacaaacccgca ttcaacaactggagtatagactggctgagaccttgcgcgcccagctctccgcaactcgcgagaggttcggaaatgccgtaacgcatttggagg ccgtgagcccactgtcaaccctcgctcggggctactccgtgacgactgccacggacggcaatgtgctcaaaaaggtaaaacaagtcaaagct ggagaaatgcttactactcggctcgaagacggatggatcgaaagtgaagtcaaaaatatacaacctgtcaagaagagtcggaaaaaggtgcat tga SEQ ID NO 30 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccccgaaaaaaaacgaagcccccgcctcctttgagaaagcacttagcgagctggagcagatcgtg acgcgcttggaatcaggggatctccctttggaagaggcattgaatgagtttgagcgaggagttcagctcgctagacaaggccaggccaaactt caacaggcggaacagcgagtccagattctccttagtgataatgaggatgcctctctgacaccgttcacgccagacaacgagtga 3xFlag_NLS_ XseB Escherichia coli SEQ ID NO 31 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcacc gacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctg ttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatct gcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagc acgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggt ggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacc tgaaccccgacaacagcgacgttgacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagc ggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaag aatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcag ctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacc tgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgac gagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaa cggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgag gaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctggga gagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatc ccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcg aggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcc caagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcct gagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaa gaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatc aaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctg agccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaactt catgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcac attgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggc acaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacct gtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctca gagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaaga ggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgag agaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatc ctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccga tttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgc cctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgag caggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatcc ggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctga gcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataa gctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaag tggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccat cgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccg 3xFlag-NLS-SpCas9-NLS (Addgene#10 00000055) Streptococcus pyogenes gaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagcc actatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgag cagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccat cagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccgg aagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctc agctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaa SEQ ID NO 32 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcacc gacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctg ttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatct gcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagc acgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggt ggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacc tgaaccccgacaacagcgacgttgacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagc ggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaag aatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcag ctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacc tgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgac gagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaa cggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgag gaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctggga gagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatc ccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcg aggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcc caagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcct gagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaa gaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatc aaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctg agccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaactt catgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcac attgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggc acaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacct gtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctca gagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaaga ggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgag agaggcggcctgagcgaactggataaggccggcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcct ggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgattt ccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccc tgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagca ggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccgg aagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagc atgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagc tgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtg gaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcg actttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccgga agagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccact atgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagca gatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatca gagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaa gaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcag ctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaa 3xFlag-NLS-SpCas9(ΔF91 6)-NLS Streptococcus pyogenes SEQ ID NO 33 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcacc gacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctg 3xFlag-NLS-SpCas9(G915 F)-NLS ttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatct gcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagc acgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggt ggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacc tgaaccccgacaacagcgacgttgacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagc ggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaag aatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcag ctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacc tgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgac gagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaa cggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgag gaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctggga gagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatc ccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcg aggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcc caagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcct gagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaa gaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatc aaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctg agccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaactt catgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcac attgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggc acaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacct gtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctca gagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaaga ggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgag agaggcggcctgagcgaactggataaggcctttttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcct ggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgattt ccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccc tgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagca ggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccgg aagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagc atgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagc tgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtg gaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcg actttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccgga agagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccact atgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagca gatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatca gagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaa gaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcag ctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaa Streptococcus pyogenes SEQ ID NO 34 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcacc gacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctg ttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatct gcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagc acgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggt ggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacc tgaaccccgacaacagcgacgttgacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagc ggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaag aatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcag ctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacc tgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgac 3xFlag-NLS-SpCas9(Q920 P)-NLS Streptococcus pyogenes gagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaa cggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgag gaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctggga gagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatc ccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcg aggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcc caagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcct gagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaa gaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatc aaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctg agccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaactt catgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcac attgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggc acaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacct gtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctca gagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaaga ggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgag agaggcggcctgagcgaactggataaggccggcttcatcaagagacccctggtggaaacccggcagatcacaaagcacgtggcacagatc ctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccga tttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgc cctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgag caggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatcc ggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctga gcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataa gctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaag tggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccat cgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccg gaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagcc actatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgag cagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccat cagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccgg aagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctc agctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaa SEQ ID NO 35 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcacc gacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctg ttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatct gcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagc acgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggt ggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacc tgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagc ggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaag aatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcag ctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacc tgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagc gcctctatgatcaagagatacgac gagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaa cggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgag gaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctggga gagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatc ccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcg aggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcc caagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcct gagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaa gaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatc 3xFlag-NLS-SpCas9(F916 P)-NLS: Streptococcus pyogenes aaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctg agccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaactt catgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcac attgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggc acaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacct gtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctca gagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaaga ggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgag agaggcggcctgagcgaactggataaggccggccccatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagat cctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccg atttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccg ccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcga gcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatc cggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctg agcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgata agctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaa gtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatccca tcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggcc ggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagc cactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcga gcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagccca tcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccg gaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtct cagctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaa SEQ ID NO 36 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcacc gacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctg ttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatct gcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagc acgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggt ggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacc tgaaccccgacaacagcgacgttgacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagc ggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaag aatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcag ctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacc tgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagc gcctctatgatcaagagatacgac gagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaa cggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgag gaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctggga gagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatc ccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcg aggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcc caagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcct gagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaa gaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatc aaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctg agccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaactt catgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcac attgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggc acaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacct gtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctca gagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaaga 3xFlag-NLS-SpCas9(R918 A) -NLS Streptococcus pyogenes SEQ ID NO SEQUENCE ggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgag agaggcggcctgagcgaactggataaggccggcttcatcgccagacagctggtggaaacccggcagatcacaaagcacgtggcacagatc ctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccga tttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgc cctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgag caggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatcc ggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctga gcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataa gctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaag tggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccat cgactttctggaagccaagggctacaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccg gaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagcc actatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgag cagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccat cagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccgg aagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctc agctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaa SEQ ID NO 37 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcacc gacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctg ttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatct gcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagc acgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggt ggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacc tgaaccccgacaacagcgacgttgacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagc ggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaag aatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcag ctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacc tgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagc gcctctatgatcaagagatacgac gagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaa cggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgag gaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctggga gagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatc ccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcg aggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcc caagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcct gagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaa gaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatc aaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctg agccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaactt catgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcac attgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggc acaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacct gtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctca gagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaaga ggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgag agaggcggcctgagcgaactggataaggccggcttcatcaagccccagctggtggaaacccggcagatcacaaagcacgtggcacagatc ctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccga tttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgc cctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgag caggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatcc ggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctga gcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataa gctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaag 3xFlag-NLS-SpCas9(R919 P)-NLS Streptococcus pyogenes tggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccat cgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccg gaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagcc actatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgag cagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccat cagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccgg aagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctc agctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaa SEQ ID NO 38 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaa ggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcacc gacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctg ttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatct gcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagc acgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggt ggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacc tgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagc ggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaag aatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcag ctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacc tgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagc gcctctatgatcaagagatacgac gagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaa cggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgag gaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctggga gagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatc ccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcg aggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcc caagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcct gagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaa gaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatc aaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctg agccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgcctgcagaaactt catgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcac attgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggc acaagcccgagaacatcgtgatcgaaatggccagagagaaccagatcacccagaagggacagaagaacagccgcgagagaatgaagcg gatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacct gtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctca gagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaaga ggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgag agaggcggcctgagcgaactggataaggccatgttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatc ctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccga tttccggaaggatttccagttttacaaagtgcgcgagatcaacaaataccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgc cctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgag caggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatcc ggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctga gcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataa gctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaag tggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccat cgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccg gaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagcc actatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgag cagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccat cagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccgg aagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctc agctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaa 3xFlag-NLS-SpCas9-NLS(N690C/ T769I/G915 M/N980K): LZ3Cas9Add gene# 140561 Streptococcus pyogenes SEQ ID NO 39 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccgaagaaaaagcgcaa ggtcgaagcgtccatgaaaaggaactacattctggggctggacatcgggattacaagcgtggggtatgggattattgactatgaaacaaggga cgtgatcgacgcaggcgtcagactgttcaaggaggccaacgtggaaaacaatgagggacggagaagcaagaggggagccaggcgcctg aaacgacggagaaggcacagaatccagagggtgaagaaactgctgttcgattacaacctgctgaccgaccattctgagctgagtggaattaat ccttatgaagccagggtgaaaggcctgagtcagaagctgtcagaggaagagttttccgcagctctgctgcacctggctaagcgccgaggagt gcataacgtcaatgaggtggaagaggacaccggcaacgagctgtctacaaaggaacagatctcacgcaatagcaaagctctggaagagaa gtatgtcgcagagctacagctggaacggctgaagaaagatggcgaggtgagagggtcaattaataggttcaagacaagcgactacgtcaaag aagccaagcagctgctgaaagtgcagaaggcttaccaccagctggatcagagcttcatcgatacttatatcgacctgctggagactcggagaa cctactatgagggaccaggagaagggagccccttcggatggaaagacatcaaggaatggtacgagatgctgatgggacattgcacctattttc cagaagagctgagaagcgtcaagtacgcttataacgcagatctgtacaacgccctgaatgacctgaacaacctggtcatcaccagggatgaaa acgagaaactggaatactatgagaagttccagatcatcgaaaacgtgtttaagcagaagaaaaagcctacactgaaacagattgctaaggaga tcctggtcaacgaagaggacatcaagggctaccgggtgacaagcactggaaaaccagagttcaccaatctgaaagtgtatcacgatattaagg acatcacagcacggaaagaaatcattgagaacgccgaactgctggatcagattgctaagatcctgactatctaccagagttccgaggacatcca ggaagagctgactaacctgaacagcgagctgacccaggaagagatcgaacagattagtaatctgaaggggtacaccggaacacacaacctg tccctgaaagctatcaatctgattctggatgagctgtggcatacaaacgacaatcagattgcaatctttaaccggctgaagctggtaccaaaaaa ggtggacctgagtcagcagaaagagatcccaaccacactggtggacgatttcattctgtcacccgtggtcaagcggagcttcatccagagcat caaagtgatcaacgccatcatcaagaagtacggcctgcccaatgatatcattatcgagctggctagggagaagaacagcaaggacgcacaga agatgatcaatgagatgcagaaacgaaaccggcagaccaatgaacgcattgaagagattatccgaactaccgggaaagagaacgcaaagta cctgattgaaaaaatcaagctgcacgatatgcaggagggaaagtgtctgtattctctggaggccatccccctggaggacctgctgaacaatcca ttcaactacgaggtcgatcatattatccccagaagcgtgtccttcgacaattcctttaacaacaaggtgctggtcaagcaggaagagaactctaaa aagggcaataggactcctttccagtacctgtctagttcagattccaagatctcttacgaaacctttaaaaagcacattctgaatctggccaaaggaa agggccgcatcagcaagaccaaaaaggagtacctgctggaagagcgggacatcaacagattctccgtccagaaggattttattaaccggaat ctggtggacacaagatacgctactcgcggcctgatgaatctgctgcgatcctatttccgggtgaacaatctggatgtgaaagtcaagtccatcaa cggcgggttcacatcttttctgaggcgcaaatggaagtttaaaaaggagcgcaacaaagggtacaagcaccatgccgaagatgctctgattatc gcaaatgccgacttcatctttaaggagtggaaaaagctggacaaagccaagaaagtgatggagaaccagatgttcgaagagaagcaggccg aatctatgcccgaaatcgagacagaacaggagtacaaggagattttcatcactcctcaccagatcaagcatatcaaggatttcaaggactacaa gtactctcaccgggtggataaaaagcccaacagagagctgatcaatgacaccctgtatagtacaagaaaagacgataaggggaataccctgat tgtgaacaatctgaacggactgtacgacaaagataatgacaagctgaaaaagctgatcaacaaaagtcccgagaagctgctgatgtaccacca tgatcctcagacatatcagaaactgaagctgattatggagcagtacggcgacgagaagaacccactgtataagtactatgaagagactgggaa ctacctgaccaagtatagcaaaaaggataatggccccgtgatcaagaagatcaagtactatgggaacaagctgaatgcccatctggacatcac agacgattaccctaacagtcgcaacaaggtggtcaagctgtcactgaagccatacagattcgatgtctatctggacaacggcgtgtataaatttgt gactgtcaagaatctggatgtcatcaaaaaggagaactactatgaagtgaatagcaagtgctacgaagaggctaaaaagctgaaaaagattag caaccaggcagagttcatcgcctccttttacaacaacgacctgattaagatcaatggcgaactgtatagggtcatcggggtgaacaatgatctgc tgaaccgcattgaagtgaatatgattgacatcacttaccgagagtatctggaaaacatgaatgataagcgcccccctcgaattatcaaaacaatc gcctctaagactcagagtatcaaaaagtactcaaccgacattctgggaaacctgtatgaggtgaagagcaaaaagcaccctcagattatcaaaa agggcaggtccggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggcccaatggtgagcaagggc gaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgag ggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgac ctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagc gcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaa gggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaag cagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacc cccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgat cacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa 3xFlag-NLS-SaCas9-P2A-EGFP Staphylococc us aureus SEQ ID NO 40 atgagcatctaccaggagttcgtcaacaagtattcactgagtaagacactgcggttcgagctgatcccacagggcaagacactggagaacatc aaggcccgaggcctgattctggacgatgagaagcgggcaaaagactataagaaagccaagcagatcattgataaataccaccagttctttatc gaggaaattctgagctccgtgtgcatcagtgaggatctgctgcagaattactcagacgtgtacttcaagctgaagaagagcgacgatgacaacc tgcagaaggacttcaagtccgccaaggacaccatcaagaaacagattagcgagtacatcaaggactccgaaaagtttaaaaatctgttcaacc agaatctgatcgatgctaagaaaggccaggagtccgacctgatcctgtggctgaaacagtctaaggacaatgggattgaactgttcaaggctaa ctccgatatcactgatattgacgaggcactggaaatcatcaagagcttcaagggatggaccacatactttaaaggcttccacgagaaccgcaag aacgtgtactccagcaacgacattcctacctccatcatctaccgaatcgtcgatgacaatctgccaaagttcctggagaacaaggccaaatatga atctctgaaggacaaagctcccgaggcaattaattacgaacagatcaagaaagatctggctgaggaactgacattcgatatcgactataagact agcgaggtgaaccagagggtcttttccctggacgaggtgtttgaaatcgccaatttcaacaattacctgaaccagtccggcattactaaattcaat accatcattggcgggaagtttgtgaacggggagaataccaagcgcaagggaattaacgaatacatcaatctgtatagccagcagatcaacgac aaaactctgaagaaatacaagatgtctgtgctgttcaaacagatcctgagtgataccgagtccaagtcttttgtcattgataaactggaagatgact cagacgtggtcactaccatgcagagcttttatgagcagatcgccgctttcaagacagtggaggaaaaatctattaaggaaactctgagtctgctg FnCas12a-NLS-3xHA(addgen e#64709) Francisella novicida ttcgatgacctgaaagcccagaagctggacctgagtaagatctacttcaaaaacgataagagtctgacagacctgtcacagcaggtgtttgatg actattccgtgattgggaccgccgtcctggagtacattacacagcagatcgctccaaagaacctggataatccctctaagaaagagcaggaact gatcgctaagaaaaccgagaaggcaaaatatctgagtctggaaacaattaagctggcactggaggagttcaacaagcacagggatattgaca aacagtgccgctttgaggaaatcctggccaacttcgcagccatccccatgatttttgatgagatcgcccagaacaaagacaatctggctcagatc agtattaagtaccagaaccagggcaagaaagacctgctgcaggcttcagcagaagatgacgtgaaagccatcaaggatctgctggaccaga ccaacaatctgctgcacaagctgaaaatcttccatattagtcagtcagaggataaggctaatatcctggataaagacgaacacttctacctggtgtt cgaggaatgttacttcgagctggcaaacattgtccccctgtataacaagattaggaactacatcacacagaagccttactctgacgagaagtttaa actgaacttcgaaaatagtaccctggccaacgggtgggataagaacaaggagcctgacaacacagctatcctgttcatcaaggatgacaagta ctatctgggagtgatgaataagaaaaacaataagatcttcgatgacaaagccattaaggagaacaaaggggaaggatacaagaaaatcgtgta taagctgctgcccggcgcaaataagatgctgcctaaggtgttcttcagcgccaagagtatcaaattctacaacccatccgaggacatcctgcgg attagaaatcactcaacacatactaagaacgggagcccccagaagggatatgagaaatttgagttcaacatcgaggattgcaggaagtttattg acttctacaagcagagcatctccaaacaccctgaatggaaggattttggcttccggttttccgacacacagagatataactctatcgacgagttcta ccgcgaggtggaaaatcaggggtataagctgacttttgagaacatttctgaaagttacatcgacagcgtggtcaatcagggaaagctgtacctgt tccagatctataacaaagatttttcagcatacagcaagggcagaccaaacctgcatacactgtactggaaggccctgttcgatgagaggaatctg caggacgtggtctataaactgaacggagaggccgaactgttttaccggaagcagtctattcctaagaaaatcactcacccagctaaggaggcc atcgctaacaagaacaaggacaatcctaagaaagagagcgtgttcgaatacgatctgattaaggacaagcggttcaccgaagataagttcttttt ccattgtccaatcaccattaacttcaagtcaagcggcgctaacaagttcaacgacgagatcaatctgctgctgaaggaaaaagcaaacgatgtg cacatcctgagcattgaccgaggagagcggcatctggcctactataccctggtggatggcaaagggaatatcattaagcaggatacattcaac atcattggcaatgaccggatgaaaaccaactaccacgataaactggctgcaatcgagaaggatagagactcagctaggaaggactggaagaa aatcaacaacattaaggagatgaaggaaggctatctgagccaggtggtccatgagattgcaaagctggtcatcgaatacaatgccattgtggtg ttcgaggatctgaacttcggctttaagagggggcgctttaaggtggaaaaacaggtctatcagaagctggagaaaatgctgatcgaaaagctga attacctggtgtttaaagataacgagttcgacaagaccggaggcgtcctgagagcctaccagctgacagctccctttgaaactttcaagaaaatg ggaaaacagacaggcatcatctactatgtgccagccggattcacttccaagatctgccccgtgaccggctttgtcaaccagctgtaccctaaata tgagtcagtgagcaagtcccaggaatttttcagcaagttcgataagatctgttataatctggacaaggggtacttcgagttttccttcgattacaaga acttcggcgacaaggccgctaaggggaaatggaccattgcctccttcggatctcgcctgatcaactttcgaaattccgataaaaaccacaattgg gacactagggaggtgtacccaaccaaggagctggaaaagctgctgaaagactactctatcgagtatggacatggcgaatgcatcaaggcagc catctgtggcgagagtgataagaaatttttcgccaagctgacctcagtgctgaatacaatcctgcagatgcggaactcaaagaccgggacaga actggactatctgattagccccgtggctgatgtcaacggaaacttcttcgacagcagacaggcacccaaaaatatgcctcaggatgcagacgc caacggggcctaccacatcgggctgaagggactgatgctgctgggccggatcaagaacaatcaggaggggaagaagctgaacctggtcatt aagaacgaggaatacttcgagtttgtccagaatagaaataacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaag ggatcctacccatacgatgttccagattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgactatgcctaa SEQ ID NO 41 atgacacagttcgagggctttaccaacctgtatcaggtgagcaagacactgcggtttgagctgatcccacagggcaagaccctgaagcacatc caggagcagggcttcatcgaggaggacaaggcccgcaatgatcactacaaggagctgaagcccatcatcgatcggatctacaagacctatg ccgaccagtgcctgcagctggtgcagctggattgggagaacctgagcgccgccatcgactcctatagaaaggagaaaaccgaggagacaa ggaacgccctgatcgaggagcaggccacatatcgcaatgccatccacgactacttcatcggccggacagacaacctgaccgatgccatcaat aagagacacgccgagatctacaagggcctgttcaaggccgagctgtttaatggcaaggtgctgaagcagctgggcaccgtgaccacaaccg agcacgagaacgccctgctgcggagcttcgacaagtttacaacctacttctccggcttttatgagaacaggaagaacgtgttcagcgccgagg atatcagcacagccatcccacaccgcatcgtgcaggacaacttccccaagtttaaggagaattgtcacatcttcacacgcctgatcaccgccgt gcccagcctgcgggagcactttgagaacgtgaagaaggccatcggcatcttcgtgagcacctccatcgaggaggtgttttccttccctttttataa ccagctgctgacacagacccagatcgacctgtataaccagctgctgggaggaatctctcgggaggcaggcaccgagaagatcaagggcctg aacgaggtgctgaatctggccatccagaagaatgatgagacagcccacatcatcgcctccctgccacacagattcatccccctgtttaagcaga tcctgtccgataggaacaccctgtctttcatcctggaggagtttaagagcgacgaggaagtgatccagtccttctgcaagtacaagacactgctg agaaacgagaacgtgctggagacagccgaggccctgtttaacgagctgaacagcatcgacctgacacacatcttcatcagccacaagaagct ggagacaatcagcagcgccctgtgcgaccactgggatacactgaggaatgccctgtatgagcggagaatctccgagctgacaggcaagatc accaagtctgccaaggagaaggtgcagcgcagcctgaagcacgaggatatcaacctgcaggagatcatctctgccgcaggcaaggagctg agcgaggccttcaagcagaaaaccagcgagatcctgtcccacgcacacgccgccctggatcagccactgcctacaaccctgaagaagcag gaggagaaggagatcctgaagtctcagctggacagcctgctgggcctgtaccacctgctggactggtttgccgtggatgagtccaacgaggt ggaccccgagttctctgcccggctgaccggcatcaagctggagatggagccttctctgagcttctacaacaaggccagaaattatgccaccaa gaagccctactccgtggagaagttcaagctgaactttcagatgcctacactggcctctggctgggacgtgaataaggagaagaacaatggcgc catcctgtttgtgaagaacggcctgtactatctgggcatcatgccaaagcagaagggcaggtataaggccctgagcttcgagcccacagagaa aaccagcgagggctttgataagatgtactatgactacttccctgatgccgccaagatgatcccaaagtgcagcacccagctgaaggccgtgac agcccactttcagacccacacaacccccatcctgctgtccaacaatttcatcgagcctctggagatcacaaaggagatctacgacctgaacaat cctgagaaggagccaaagaagtttcagacagcctacgccaagaaaaccggcgaccagaagggctacagagaggccctgtgcaagtggatc gacttcacaagggattttctgtccaagtataccaagacaacctctatcgatctgtctagcctgcggccatcctctcagtataaggacctgggcgag tactatgccgagctgaatcccctgctgtaccacatcagcttccagagaatcgccgagaaggagatcatggatgccgtggagacaggcaagct gtacctgttccagatctataacaaggactttgccaagggccaccacggcaagcctaatctgcacacactgtattggaccggcctgttttctccag AsCas12a-NLS-3xHA(addgen e#69982) Acidaminoco ccus spec agaacctggccaagacaagcatcaagctgaatggccaggccgagctgttctaccgccctaagtccaggatgaagaggatggcacaccggct gggagagaagatgctgaacaagaagctgaaggatcagaaaaccccaatccccgacaccctgtaccaggagctgtacgactatgtgaatcac agactgtcccacgacctgtctgatgaggccagggccctgctgcccaacgtgatcaccaaggaggtgtctcacgagatcatcaaggataggcg ctttaccagcgacaagttctttttccacgtgcctatcacactgaactatcaggccgccaattccccatctaagttcaaccagagggtgaatgcctac ctgaaggagcaccccgagacacctatcatcggcatcgatcggggcgagagaaacctgatctatatcacagtgatcgactccaccggcaagat cctggagcagcggagcctgaacaccatccagcagtttgattaccagaagaagctggacaacagggagaaggagagggtggcagcaaggc aggcctggtctgtggtgggcacaatcaaggatctgaagcagggctatctgagccaggtcatccacgagatcgtggacctgatgatccactacc aggccgtggtggtgctggagaacctgaatttcggctttaagagcaagaggaccggcatcgccgagaaggccgtgtaccagcagttcgagaa gatgctgatcgataagctgaattgcctggtgctgaaggactatccagcagagaaagtgggaggcgtgctgaacccataccagctgacagacc agttcacctcctttgccaagatgggcacccagtctggcttcctgttttacgtgcctgccccatatacatctaagatcgatcccctgaccggcttcgt ggaccccttcgtgtggaaaaccatcaagaatcacgagagccgcaagcacttcctggagggcttcgactttctgcactacgacgtgaaaaccgg cgacttcatcctgcactttaagatgaacagaaatctgtccttccagaggggcctgcccggctttatgcctgcatgggatatcgtgttcgagaagaa cgagacacagtttgacgccaagggcacccctttcatcgccggcaagagaatcgtgccagtgatcgagaatcacagattcaccggcagatacc gggacctgtatcctgccaacgagctgatcgccctgctggaggagaagggcatcgtgttcagggatggctccaacatcctgccaaagctgctg gagaatgacgattctcacgccatcgacaccatggtggccctgatccgcagcgtgctgcagatgcggaactccaatgccgccacaggcgagg actatatcaacagccccgtgcgcgatctgaatggcgtgtgcttcgactcccggtttcagaacccagagtggcccatggacgccgatgccaatg gcgcctaccacatcgccctgaagggccagctgctgctgaatcacctgaaggagagcaaggatctgaagctgcagaacggcatctccaatca ggactggctggcctacatccaggagctgcgcaacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatcc tacccatacgatgttccagattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgactatgcctaa SEQ ID NO 42 atgagcaagctggagaagtttacaaactgctactccctgtctaagaccctgaggttcaaggccatccctgtgggcaagacccaggagaacatc gacaataagcggctgctggtggaggacgagaagagagccgaggattataagggcgtgaagaagctgctggatcgctactatctgtcttttatc aacgacgtgctgcacagcatcaagctgaagaatctgaacaattacatcagcctgttccggaagaaaaccagaaccgagaaggagaataagg agctggagaacctggagatcaatctgcggaaggagatcgccaaggccttcaagggcaacgagggctacaagtccctgtttaagaaggatatc atcgagacaatcctgccagagttcctggacgataaggacgagatcgccctggtgaacagcttcaatggctttaccacagccttcaccggcttctt tgataacagagagaatatgttttccgaggaggccaagagcacatccatcgccttcaggtgtatcaacgagaatctgacccgctacatctctaata tggacatcttcgagaaggtggacgccatctttgataagcacgaggtgcaggagatcaaggagaagatcctgaacagcgactatgatgtggag gatttctttgagggcgagttctttaactttgtgctgacacaggagggcatcgacgtgtataacgccatcatcggcggcttcgtgaccgagagcgg cgagaagatcaagggcctgaacgagtacatcaacctgtataatcagaaaaccaagcagaagctgcctaagtttaagccactgtataagcaggt gctgagcgatcgggagtctctgagcttctacggcgagggctatacatccgatgaggaggtgctggaggtgtttagaaacaccctgaacaagaa cagcgagatcttcagctccatcaagaagctggagaagctgttcaagaattttgacgagtactctagcgccggcatctttgtgaagaacggcccc gccatcagcacaatctccaaggatatcttcggcgagtggaacgtgatccgggacaagtggaatgccgagtatgacgatatccacctgaagaa gaaggccgtggtgaccgagaagtacgaggacgatcggagaaagtccttcaagaagatcggctccttttctctggagcagctgcaggagtacg ccgacgccgatctgtctgtggtggagaagctgaaggagatcatcatccagaaggtggatgagatctacaaggtgtatggctcctctgagaagc tgttcgacgccgattttgtgctggagaagagcctgaagaagaacgacgccgtggtggccatcatgaaggacctgctggattctgtgaagagctt cgagaattacatcaaggccttctttggcgagggcaaggagacaaacagggacgagtccttctatggcgattttgtgctggcctacgacatcctg ctgaaggtggaccacatctacgatgccatccgcaattatgtgacccagaagccctactctaaggataagttcaagctgtattttcagaaccctcag ttcatgggcggctgggacaaggataaggagacagactatcgggccaccatcctgagatacggctccaagtactatctggccatcatggataag aagtacgccaagtgcctgcagaagatcgacaaggacgatgtgaacggcaattacgagaagatcaactataagctgctgcccggccctaataa gatgctgccaaaggtgttcttttctaagaagtggatggcctactataaccccagcgaggacatccagaagatctacaagaatggcacattcaag aagggcgatatgtttaacctgaatgactgtcacaagctgatcgacttctttaaggatagcatctcccggtatccaaagtggtccaatgcctacgatt tcaacttttctgagacagagaagtataaggacatcgccggcttttacagagaggtggaggagcagggctataaggtgagcttcgagtctgcca gcaagaaggaggtggataagctggtggaggagggcaagctgtatatgttccagatctataacaaggacttttccgataagtctcacggcacac ccaatctgcacaccatgtacttcaagctgctgtttgacgagaacaatcacggacagatcaggctgagcggaggagcagagctgttcatgaggc gcgcctccctgaagaaggaggagctggtggtgcacccagccaactcccctatcgccaacaagaatccagataatcccaagaaaaccacaac cctgtcctacgacgtgtataaggataagaggttttctgaggaccagtacgagctgcacatcccaatcgccatcaataagtgccccaagaacatct tcaagatcaatacagaggtgcgcgtgctgctgaagcacgacgataacccctatgtgatcggcatcgataggggcgagcgcaatctgctgtata tcgtggtggtggacggcaagggcaacatcgtggagcagtattccctgaacgagatcatcaacaacttcaacggcatcaggatcaagacagatt accactctctgctggacaagaaggagaaggagaggttcgaggcccgccagaactggacctccatcgagaatatcaaggagctgaaggccg gctatatctctcaggtggtgcacaagatctgcgagctggtggagaagtacgatgccgtgatcgccctggaggacctgaactctggctttaagaa tagccgcgtgaaggtggagaagcaggtgtatcagaagttcgagaagatgctgatcgataagctgaactacatggtggacaagaagtctaatcc ttgtgcaacaggcggcgccctgaagggctatcagatcaccaataagttcgagagctttaagtccatgtctacccagaacggcttcatcttttacat ccctgcctggctgacatccaagatcgatccatctaccggctttgtgaacctgctgaaaaccaagtataccagcatcgccgattccaagaagttca tcagctcctttgacaggatcatgtacgtgcccgaggaggatctgttcgagtttgccctggactataagaacttctctcgcacagacgccgattaca tcaagaagtggaagctgtactcctacggcaaccggatcagaatcttccggaatcctaagaagaacaacgtgttcgactgggaggaggtgtgcc tgaccagcgcctataaggagctgttcaacaagtacggcatcaattatcagcagggcgatatcagagccctgctgtgcgagcagtccgacaag gccttctactctagctttatggccctgatgagcctgatgctgcagatgcggaacagcatcacaggccgcaccgacgtggattttctgatcagccc HLbCas12a-NLS-3xHA(addgen e#69988) Lachnospirac eae bacterium tgtgaagaactccgacggcatcttctacgatagccggaactatgaggcccaggagaatgccatcctgccaaagaacgccgacgccaatggc gcctataacatcgccagaaaggtgctgtgggccatcggccagttcaagaaggccgaggacgagaagctggataaggtgaagatcgccatct ctaacaaggagtggctggagtacgcccagaccagcgtgaagcacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaa aaagggatcctacccatacgatgttccagattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgactatgcctaa SEQ ID NO 43 3xGS ggcggcgggagcgggggtggcagcggcggcgggtcg SEQ ID NO 44 tctggaggatctagcggaggatcctctggcagcgagacaccaggaacaagcgagtcagcaacaccagagagcagtggcggcagcagcgg cggctcg (SGGS)2-XTEN-(SGGS)2 SEQ ID NO 45 gctgaggcggcggcaaaagaagcagcggcaaaagaagctgccgcaaaggaagcagcagcaaaagcccttgaagccgaagctgctgcta aggaggctgccgcaaaagaggctgccgccaaagaagcagccgctaaagcg A(EAAAK)4 A SEQ ID NO 46 ggatccgactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcg gaaggtcggtatccacggagtcccagcagcg 3xFlag-NLS SEQ ID NO 47 ggatccgactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagggtatccacggagtcccagc agcg 3xFlag SEQ ID NO 48 GGTTCTGGAAGTGAGGCAGCTGCGAAGGAGGCTGCGGCGAAAGAAGCTGCAGCAAA GGAAGCAGCAGCAAAGGCACTGGAGGCCGCTGCTGCTAAAGAGGCTGCCGCCAAAG AAGCTGCGGCAAAGGAAGCTGCGGCTAAGGGAAGTGGGAGCGCAGCGGCCAAAGAG GCAGCGGCCAAGGAAGCTGCTGCAAAAGAAGCAGCAGCTAAAGGGAGCGGATCG (H4)3: SEQ ID NO 49 GCCGGAAGCGGTGGTTCAGGGGGATCCGGAGGAAGTCCTGTTCCCTCTACCCCACCA ACTAATAGCAGCTCAACCCCTCCGACCCCCTCTCCGTCACCGGTGCCGAGTACCCCGC CAACCAATAGCTCATCAACTCCGCCTACGCCGTCCCCTAGTCCAGTACCTAGCACCCC TCCAACAAATTCTAGCAGTACACCACCCACACCAAGCCCTAGCGCGTCG GPcPcPc SEQ ID NO 50 GCTGGTTCTGGTGGCTCAGGGGGTTCCGGTGGTTCCCCAGTACCAAGTACTCCTCCCA CTCCCTCTCCAAGTACGCCGCCTACACCCTCACCCAGCGGCGGCTCTGGCAATTCCAG TGGTTCAGGCGGTAGTCCCGTGCCAAGTACGCCACCAACTCCAAGTCCATCAACACC ACCGACCCCTTCTCCGTCTGCATCG GPGcP SEQ ID NO 51 GCGGGTTCTGGAGGTTCAGGCGGGAGCGGTGGCAGTCCAGTGCCGAGCACACCGCCA ACACCGAGCCCAAGTACGCCACCGACTCCAAGTCCCAGCATACAGCGAACACCGAAG ATTCAGGTCTACTCACGACACCCAGCGGAAAACGGCAAATCTAATTTTCTGAATTGCT ATGTTTCCGGTTTTCACCCCTCAGACATCGAGGTCGACCTGCTGAAGAACGGTGAAAG GATTGAAAAGGTTGAACACTCCGACTTGAGCTTTAGTAAGGACTGGTCATTCTATTTG CTGTATTACACCGAGTTCACTCCGACCGAAAAGGATGAATACGCATGTCGAGTGAAT CATGTCACGCTGAGCCAACCCAAGATCGTGAAATGGGACAGGGACGGGGGGTCTGG GGGTAGCGGAGGAAGCGGCGGGTCTATCCAACGCACTCCAAAAATTCAAGTCTACTC AAGACACCCTGCCGAGAATGGAAAATCAAACTTTTTGAATTGCTACGTCTCTGGATTC CATCCGTCAGACATCGAAGTTGATCTGTTGAAAAACGGTGAGCGAATTGAGAAAGTG GAGCATTCAGATCTTAGCTTCAGTAAAGACTGGTCCTTTTATCTCTTGTATTACACGG AGTTCACTCCCACAGAAAAAGATGAATACGCCTGTCGAGTTAACCACGTCACGCTGT CACAGCCAAAGATAGTGAAATGGGATCGCGACCCAGTGCCCTCAACACCCCCTACTC CTAGTCCGAGCACTCCTCCAACGCCTTCACCATCTGCCTCG GPbGbP SEQ ID NO 52 GCTGGTTCCGGCGGATCTGGTGGATCTGGTGGCAGCCCCGTCCCTTCTACTCCACCCA CACCGTCCCCGTCAACTCCTCCCACCCCGTCTCCGTCCGATGGAAGGTACTCTCTCAC GTACATCTACACTGGGTTGTCAAAGCATGTGGAAGACGTGCCAGCCTTCCAGGCGCTT GPZP GGAAGCCTCAATGACCTTCAGTTTTTTCGCTACAATAGCAAGGATCGAAAGTCACAA CCTATGGGTCTCTGGAGACAGGTCGAAGGGATGGAGGACTGGAAACAGGATAGCCA ATTGCAAAAAGCGAGAGAGGATATCTTTATGGAGACGCTTAAAGACATTGTTGAGTA TTACAACGACTCTAACGGTAGTCACGTATTGCAGGGCCGATTTGGGTGTGAGATAGA GAATAACCGGAGTTCCGGCGCTTTTTGGAAATATTATTACGATGGCAAGGACTACATC GAGTTTAACAAAGAAATTCCAGCCTGGGTGCCTTTTGACCCAGCTGCACAAATTACA AAACAGAAGTGGGAGGCGGAGCCAGTGTACGTTCAAAGGGCAAAAGCATACTTGGA GGAAGAGTGTCCCGCAACTCTCCGAAAGTACTTGAAGTATTCTAAAAACATACTGGA TCGACAGGATCCCCCTTCAGTAGTCGTAACCTCCCACCAGGCCCCAGGTGAGAAGAA GAAGTTGAAATGCCTTGCTTACGACTTCTACCCAGGCAAGATTGATGTTCACTGGACA AGGGCTGGTGAGGTCCAAGAGCCCGAACTTAGAGGGGATGTGTTGCATAACGGTAAT GGGACGTATCAGTCATGGGTCGTGGTGGCAGTCCCTCCTCAAGATACGGCACCATAC TCTTGCCATGTGCAACACAGCTCACTGGCGCAGCCACTCGTAGTGCCTTGGGAGGCCA GCCCCGTGCCATCAACTCCCCCAACTCCATCACCTAGTACCCCCCCTACTCCGTCAGC CTCG SEQ ID NO 53 GCTGGTTCTGGGGGGTCAGGAGGGAGTGGAGGGTCTGGAGGTTCTGGAGGCTCAGGA GGTAGCGGTGGTAGTGACGGCAGGTACAGTCTCACCTATATCTATACAGGATTGTCTA AGCATGTTGAAGACGTGCCCGCCTTTCAGGCACTGGGTTCTTTGAACGACCTCCAGTT TTTCCGCTACAACAGTAAAGACCGAAAATCTCAGCCCATGGGGCTCTGGAGACAAGT TGAAGGTATGGAGGACTGGAAACAGGACAGTCAATTGCAAAAGGCCAGAGAAGATA TTTTTATGGAAACCTTGAAGGATATTGTCGAGTACTACAACGATTCAAACGGGTCCCA CGTGCTGCAGGGCCGATTCGGTTGCGAGATAGAAAATAATCGATCTAGTGGTGCCTTT TGGAAGTATTACTACGACGGAAAAGATTATATCGAATTTAATAAAGAGATTCCTGCG TGGGTGCCGTTTGACCCGGCGGCACAAATTACTAAACAAAAGTGGGAAGCGGAACCG GTGTATGTTCAGAGGGCTAAGGCGTACCTTGAAGAAGAGTGCCCCGCTACGTTGAGG AAATACCTCAAATATTCCAAAAATATCTTGGATCGACAAGATCCACCTAGCGTGGTTG TTACTTCACACCAAGCACCAGGTGAAAAAAAAAAATTGAAGTGTCTTGCATATGACT TCTATCCTGGGAAGATCGACGTACACTGGACACGAGCCGGAGAGGTACAAGAACCTG AACTGCGAGGGGACGTCCTCCATAACGGGAACGGTACCTATCAAAGTTGGGTGGTGG TTGCGGTTCCACCTCAGGACACTGCGCCTTACTCCTGTCACGTGCAGCATTCCTCTCTC GCTCAACCCCTTGTCGTGCCGTGGGAGGCCTCCGGAGGGTCTGGCGGAAGCGGAGGA TCTGGTGGGTCCGATGGTAGGTACTCACTTACTTACATATACACGGGTCTTAGTAAAC ACGTCGAGGATGTCCCGGCGTTCCAAGCTCTGGGTAGTTTGAATGATCTCCAATTTTT TAGATACAATAGCAAAGATCGAAAAAGCCAACCAATGGGACTCTGGAGACAGGTGG AGGGAATGGAAGATTGGAAACAAGATTCTCAACTCCAGAAGGCTAGGGAAGACATTT TCATGGAAACGCTCAAAGATATTGTAGAGTATTATAATGATTCTAACGGCAGCCACG TCCTTCAGGGGCGATTTGGGTGTGAGATTGAAAACAATCGATCTAGCGGTGCATTTTG GAAATATTACTATGATGGCAAAGACTATATCGAATTCAACAAGGAAATTCCAGCATG GGTCCCATTCGACCCCGCGGCTCAAATTACCAAGCAAAAATGGGAAGCCGAACCTGT CTACGTACAACGGGCGAAGGCATATCTTGAGGAGGAATGCCCCGCGACCCTCCGAAA GTACCTTAAGTACTCCAAGAACATTCTCGATCGGCAGGACCCCCCTTCTGTGGTAGTC ACCAGCCATCAGGCACCTGGGGAGAAGAAGAAACTCAAGTGCCTGGCCTACGATTTC TACCCTGGGAAAATCGATGTCCACTGGACGAGAGCGGGTGAGGTGCAAGAGCCAGA ATTGAGAGGTGATGTCCTTCATAACGGCAATGGCACCTATCAGTCATGGGTGGTCGTG GCTGTTCCCCCTCAAGACACGGCACCGTATAGCTGTCATGTCCAACACTCCTCCCTCG CTCAACCACTCGTGGTCCCATGGGAGGCTAGCCCAGTGCCCAGCACACCCCCTACTCC CTCTCCTTCTACTCCACCGACCCCTTCACCGTCCGCTTCG GGZGZP SEQ ID NO 54 GGGCTGAGAGAGGGACAAGTgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgg caccgagtcggtgc GGGCTGAG AGAGGGAC AAGT SEQ ID NO 55 AGTGTGCATTGCCACCTCAGgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggc accgagtcggtgc AGTGTGCA TTGCCACC TCAG SEQ ID NO 56 GCAGGACTCCTTTCCTCCATgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggca ccgagtcggtgc GCAGGACT CCTTTCCT CCAT SEQ ID NO 57 ATAGGAGAAGATGATGTATAgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgg caccgagtcggtgc ATAGGAGA AGATGATG TATA SEQ ID NO 58 AAAACGTTTCCAAGACATGAgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggc accgagtcggtgc AAAACGTT TCCAAGAC ATGA SEQ ID NO 59 CCGCCGTCCAAGACCTACCGgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggc accgagtcggtgc CCGCCGTC CAAGACCT ACCG SEQ ID NO 60 CCAAGAAGCGCACCACCTCCgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggc accgagtcggtgc CCAAGAAG CGCACCAC CTCC SEQ ID NO 61 AGCCTGGAAGCACGAATGGTgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtg gcaccgagtcggtgc AGCCTGGA AGCACGA ATGGT SEQ ID NO 62 ACATACCAAGAGAATCACCCgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtg gcaccgagtcggtgc ACATACCA AGAGAATC ACCC: SEQ ID NO 63 GAAGGAGGAGGCCTAAGGAgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtg gcaccgagtcggtgc GAAGGAG GAGGCCTA AGGA: SEQ ID NO 64 AAGAAGACTAGCTGAGCTCTgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtg gcaccgagtcggtgc AAGAAGA CTAGCTGA GCTCT: SEQ ID NO 65 MDPRGILKAFPKRQKIHADASSKVLAKIPRREEGEEAEEWLSSLRAHVVRTGIGRARAE LFEKQIVQHGGQLCPAQGPGVTHIWDEGMDYERALRLLRLPQLPPGAQLVKSAWLSL CLQERRLVDVAGFSIFIPSRYLDHPQPSKAEQDASIPPGTHEALLQTALSPPPPPTRPVSPP QKAKEAPNTQAQPISDDEASDGEETQVSAADLEALISGHYPTSLEGDCEPSPAPAVLDK WVCAQPSSQKATNHNLHITEKLEVLAKAYSVQGDKWRALGYAKAINALKSFHKPVTS YQEACSIPGIGKRMAEKIIEILESGHLRKLDHISESVPVLELFSNIWGAGTKTAQMWYQQ GFRSLEDIRSQASLTTQQAIGLKHYSDFLERMPREEATEIEQTVQKAAQAFNSGLLCVAC GSYRRGKATCGDVDVLITHPDGRSHRGIFSRLLDSLRQEGFLTDDLVSQEENGQQQKYL GVCRLPGPGRRHRRLDIIVVPYSEFACALLYFTGSAHFNRSMRALAKTKGMSLSEHALS TAWRNTHGCKVGPGRVLPTPTEKDVFRLLGLPYREPAERDW POLL: SEQ ID NO 66 MALPKRRRARVGSPSGDAASSTPPSTRFPGVAIYLVEPRMGRSRRAFLTGLARSKGFRV LDACSSEATHVVMEETSAEEAVSWQERRMAAAPPGCTPPALLDISWLTESLGAGQPVP VECRHRLEVAGPRKGPLSPAWMPAYACQRPTPLTHHNTGLSEALEILAEAAGFEGSEGR LLTFCRAASVLKALPSPVTTLSQLQGLPHFGEHSSRWQELLEHGVCEEVERVRRSERY QTMKLFTQIFGVGVKTADRWYREGLRTLDDLREQPQKLTQQQKAGLQHHQDLSTPVL RSDVDALQQVVEEAVGQALPGATVTLTGGFRRGKLQGHDVDFLITHPKEGQEAGLLPR VMCRLQDQGLILYHQHQHSCCESPTRLAQQSHMDAFERSFCIFRLPQPPGAAVGGSTRP CPSWKAVRVDLWAPVSQFPFALLGWTGSKLFQRELRRFSRKEKGLWLNSHGLFDPEQ KTFFQAASEEDIFRHLGLEYLPPEQRNA POLM: SEQ ID NO 67 MALPKRRRARVGSPSGDAASSTPPSTRFPGVAIYLVEPRMGRSRRAFLTGLARSKGFRV LDACSSEATHVVMEETSAEEAVSWQERRMAAAPPGCTPPALLDISWLTESLGAGQPVP VECRHRLEVAGPRKGPLSPAWMPAYACQRPTPLTHHNTGLSEALEILAEAAGFEGSEGR LLTFCRAASVLKALPSPVTTLSQLQGLPHFGEHSSRWQELLEHGVCEEVERVRRSERY QTMKLFTQIFGVGVKTADRWYREGLRTLDDLREQPQKLTQQQKAGLQHHQDLSTPVL RSDVDALQQVVEEAVGQALPGATVTLTGGFRRGKLQGGDVDFLITHPKEGQEAGLLPR VMCRLQDQGLILYHQHQHSCCESPTRLAQQSHMDAFERSKCIFRLPQPPGAAVGGSTRP CPSWKAVRVDLWAPVSQFPFALLGWTGSKLFQRELRRFSRKEKGLWLNSHGLFDPEQ KTFFQAASEEDIFRHLGLEYLPPEQRNA POLM(H329 G): SEQ ID NO 68 MALPKRRRARVGSPSGDAASSTPPSTRFPGVAIYLVEPRMGRSRRAFLTGLARSKGFRV LDACSSEATHVVMEETSAEEAVSWQERRMAAAPPGCTPPALLDISWLTESLGAGQPVP VECRHRLEVAGPRKGPLSPAWMPAYACQRPTPLTHHNTGLSEALEILAEAAGFEGSEGR LLTFCRAASVLKALPSPVTTLSQLQGLPHFGEHSSRWQELLEHGVCEEVERVRRSERY QTMKLFTQIFGVGVKTADRWYREGLRTLDDLREQPQKLTQQQKAGLQHHQDLSTPVL RSDVDALQQVVEEAVGQALPGATVTLTGGFRRGKLQGGDVDFLITHPKEGQEAGLLPR VMCRLQDQGLILYHQHQHSCCESPTRLAQQSHMDAFERSFCIFRLPQPPGAAVGGSTRP CPSWKAVRVDLWAPVSQFPFALLGWTGSKLFQRELRRFSRKEKGLWLNSHGLFDPEQ KTFFQAASEEDIFRHLGLEYLPPEQRNA POLM(H329 G,R389K): SEQ ID NO 69 MALPKRRRARVGSPSGDAASSTPPSTRFPGVAIYLVEPRMGRSRRAFLTGLARSKGFRV LDACSSEATHVVMEETSAEEAVSWQERRMAAAPPGCTPPALLDISWLTESLGAGQPIPS RYLDHPQPSKAEQDASIPPGTHEALLQTALSPPPPPTRPVSPPQKAKEAPNTQAQPISDDE ASDGEETQVSAADLEALISGHYPTSLEGDCEPSPAPAVLDKWVCAQPSSQKATNHNLHI TEKLEVLAKAYSVQGDKWRALGYAKAINALKSFHKPVTSYQEACSIPGIGKRMAEKIIEI LESGHLRKLDHISESVPVLELFSNIWGAGTKTAQMWYQQGFRSLEDIRSQASLTTQQAIG LKHYSDFLERMPREEATEIEQTVQKAAQAFNSGLLCVACGSYRRGKATCGDVDVLITHP DGRSHRGIFSRLLDSLRQEGFLTDDLVSQEENGQQQKYLGVCRLPGPGRRHRRLDIIVVP YSEFACALLYFTGSAHFNRSMRALAKTKGMSLSEHALSTAVVRNTHGCKVGPGRVLPT PTEKDVFRLLGLPYREPAERDW BRCT(POL M) POLL1: SEQ ID NO 70 MALPKRRRARVGSPSGDAASSTPPSTRFPGVAIYLVEPRMGRSRRAFLTGLARSKGFRV LDACSSEATHVVMEETSAEEAVSWQERRMAAAPPGCTPPALLDISWLTESLGAGQPVP VECRHRLEVAGPRKGPLSSSQKATNHNLHITEKLEVLAKAYSVQGDKWRALGYAKAIN ALKSFHKPVTSYQEACSIPGIGKRMAEKIIEILESGHLRKLDHISESVPVLELFSNIWGAGT BRCT(POL M)_POLL2: KTAQMWYQQGFRSLEDIRSQASLTTQQAIGLKHYSDFLERMPREEATEIEQTVQKAAQA FNSGLLCVACGSYRRGKATCGDVDVLITHPDGRSHRGIFSRLLDSLRQEGFLTDDLVSQ EENGQQQKYLGVCRLPGPGRRHRRLDIIVVPYSEFACALLYFTGSAHFNRSMRALAKTK GMSLSEHALSTAVVRNTHGCKVGPGRVLPTPTEKDVFRLLGLPYREPAERDW SEQ ID NO 71 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAAIKSIASRLRGSRRFLS GFVAGAVVGAAGAGLAALQFFRSQGAEGALTGKQPDGSAEKAVLEQFGFPLTGTEARC YTNHALSYDQAKRVPRWVLEHISKSKIMGDADRKHCKFKPDPNIPPTFSAFNEDYVGSG WSRGHMAPAGNNKFSSKAMAETFYLSNIVPQDFDNNSGYWNRIEMYCRELTERFEDV WVVSGPLTLPQTRGDGKKIVSYQVIGEDNVAVPSHLYKVILARRSSVSTEPLALGAFVV PNEAIGFQPQLTEFQVSLQDLEKLSGLVFFPHLDRTSDIRNICSVDTCKLLDFQEFTLYLS TRKIEGARSVLRLEKIMENLKNAEIEPDDYFMSRYFKKT FFT K AKEQSGTQIRKPS 3xFlag-NLS-EXOG: SEQ ID NO 72 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAPRLLPISAATLALAQLT YGWGNLGHETVAYIAQSFVASSTESFCQNILGDDSTSYLANVATWADTYKYTDAGEFS KPYHFIDAQDNPPQSCGVDYDRDCGSAGCSISAIQNYTNILLESPNGSEALNALKFVVHII GDIHQPLHDENLEAGGNGIDVTYDGETTNLHHIWDTNMPEEAAGGYSLSVAKTYADLL TERIKTGTYSSKKDSWTDGIDIKDPVSTSMIWAADANTYVCSTVLDDGLAYINSTDLSGE YYDKSQPVFEELIAKAGYRLAAWLDLIASQPS 3xFlag-NLS-nucS: SEQ ID NO 73 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAWGALGHATVAYVAQH YVSPEAASWAQGILGSSSSSYLASIASWADEYRLTSAGKWSASLHFIDAEDNPPTNCNV DYERDCGSSGCSISAIANYTQRVSDSSLSSENHAEALRFLVHFIGDMTQPLHDEAYAVG GNKINVTFDGYHDNLHSDWDTYMPQKLIGGHALSDAESWAKTLVQNIESGNYTAQAIG WIKGDNISEPITTATRWASDANALVCTVVMPHGAAALQTGDLYPTYYDSVIDTIELQIA KGGYRLANWINEI 3xFlag-NLS-NucP1: SEQ ID NO 74 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAKMKLFQTICRQLRSSK FSVESAALVAFSTSSYSCGRKKKVNPYEEVDQEKYSNLVQSVLSSRGVAQTPGSVEEDA LLCGPVSKHKLPNQGEDRRVPQNWFPIFNPERSDKPNASDPSVPLKIPLQRNVIPSVTRV LQQTMTKQQVFLLERWKQRMILELGEDGFKEYTSSFHVCDHVYMKNLARDVFLQGKR FHEALESILSPQETLKERDENLLKSGYIESVQHILKDVSGVRALESAVQHETLNYIGLLDC VAEYQGKLCVIDWKTSEKPKPFIQSTFDNPLQVVAYMGAMNHDTNYSFQVQCGLIWA YKDGSPAHPHFMDAELCSQYWTKWLLRLEEYTFKKKNQNIQKPEYSE 3xFlag-NLS-MGME1: SEQ ID NO 75 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAVKQQIQLRRREVDETA DLPAELPPLLRRLYASRGVRSAQELERSVKGMLPWQQLSGVEKAVEILYNAFREGTRIIV VGDFDADGATSTALSVLAMKSLGCSNIDYLVPNRFEDGYGLSPEVVDQAHARGAQLIV TVDNGISSHAGVEHARSLGIPVIVTDHHLPGDTLPAAEAIINPNLRDCNFPSKSLAGVGV AFYLMLALRTFLRDQGWFDERNIAIPNLAELLDLVALGTVADVVPLDANNRILTWQGM SRIRAGKCRPGIKALLEVANRDAQKLAASDLGFALGPRLNAAGRLDDMSVGVALLLCD NIGEARVLANELDALNQTRKEIEQGMQIEALTLCEKLERSRDTLPGGLAMYHPEWHQG WGILASRIKERFHRPVIAFAPAGDGTLKGSGRSIQGLHMRDALERLDTLYPGMMLKFG GHAMAAGLSLEEDKFKLFQQRFGELVTEWLDPSLLQGEWSDGPLSPAEMTMEVAQLL RDAGPWGQMFPEPLFDGHFRLLQQRLVGERHLKVMVEPVGGGPLLDGIAFNVDTALW PDNGVREVQLAYKLDINEFRGNRSLQIIIDNIWPI 3xFlag-NLS-recj: SEQ ID NO 76 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAKEFYISIETVGNNIVER YIDENGKERTREVEYLPTMFRHCKEESKYKDIYGKNCAPQKFPSMKDARDWMKRMED IGLEALGNINDFKLAYISDTYGSEIVYDRKFVRVANCDIEVTGDKFPDPMKAEYEIDAITH YDSIDDRFYVFDLLNSMYGSVSKWDAKLAAKLDCEGGDEVPQEILDRVIYMPFDNERD MLMEYINLWEQKRPAIFTGWNIEGFDVPYIMNRVKMILGERSMKRFSPIGRVKSKLIQN MYGSKEIYSIDGVSILDYLDLYKKFAFTNLPSFSLESVAQHETKKGKLPYDGPINKLRET NHQRYISYNIIDVESVQAIDKIRGFIDLVLSMSYYAKMPFSGVMSPIKTWDAIIFNSLKGE HKVIPQQGSHVKQSFPGAFVFEPKPIARRYIMSFDLTSLYPSIIRQVNISPETIRGQFKVHPI HEYIAGTAPKPSDEYSCSPNGWMYDKHQEGIIPKEIAKVFFQRKDWKKKMFAEEMNAE AIKKIIMKGAGSCSTKPEVERYVKFSDDFLNELSNYTESVLNSLIEECEKAATLANTNQL NRKILINSLYGALGNIHFRYYDLRNATAITIFGQVGIQWIARKINEYLNKVCGTNDEDFIA AGDTDSVYVCVDKVIEKVGLDRFKEQNDLVEFMNQFGKKKMEPMIDVAYRELCDYM NNREHLMHMDREAISCPPLGSKGVGGFWKAKKRYALNVYDMEDKRFAEPHLKIMGM ETQQSSTPKAVQEALEESIRRILQEGEESVQEYYKNFEKEYRQLDYKVIAEVKTANDIAK 3xFlag-NLS-T4 DNA polymerase: YDDKGWPGFKCPFHIRGVLTYRRAVSGLGVAPILDGNKVMVLPLREGNPFGDKCIAWP SGTELPKEIRSDVLSWIDHSTLFQKSFVKPLAGMCESAGMDYEEKASLDFLFG SEQ ID NO 77 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAKEFYISIETVGNNIVER YIDENGKERTREVEYLPTMFRHCKEESKYKDIYGKNCAPQKFPSMKDARDWMKRMED IGLEALGNINDFKLAYISDTYGSEIVYDRKFVRVANCDIEVTGDKFPDPMKAEYEIDAITH YDSIDDRFYVFDLLNSMYGSVSKWDAKLAAKLDCEGGDEVPQEILDRVIYMPFDNERD MLMEYINLWEQKRPAIFTGWNIEGFDVPYIMNRVKMILGERSMKRFSPIGRVKSKLIQN MYGSKEIYSIDGVSILDYLDLYKKFAFTNLPSFSLESVAQHETKKGKLPYDGPINKLRET NHQRYISANIIDVESVQAIDKIRGFIDLVLSMSYYAKMPFSGVMSPIKTWDAIIFNSLKGE HKVIPQQGSHVKQSFPGAFVFEPKPIARRYIMSFDLTSLYPSIIRQVNISPETIRGQFKVHPI HEYIAGTAPKPSDEYSCSPNGWMYDKHQEGIIPKEIAKVFFQRKDWKKKMFAEEMNAE AIKKIIMKGAGSCSTKPEVERYVKFSDDFLNELSNYTESVLNSLIEECEKAATLANTNQL NRKILINSLYGALGNIHFRYYDLRNATAITIFGQVGIQWIARKINEYLNKVCGTNDEDFIA AGDTDSVYVCVDKVIEKVGLDRFKEQNDLVEFMNQFGKKKMEPMIDVAYRELCDYM NNREHLMHMDREAISCPPLGSKGVGGFWKAKKRYALNVYDMEDKRFAEPHLKIMGM ETQQSSTPKAVQEALEESIRRILQEGEESVQEYYKNFEKEYRQLDYKVIAEVKTANDIAK YDDKGWPGFKCPFHIRGVLTYRRAVSGLGVAPILDGNKVMVLPLREGNPFGDKCIAWP SGTELPKEIRSDVLSWIDHSTLFQKSFVKPLAGMCESAGMDYEEKASLDFLFG 3xFlag-NLS-T4 DNA polymerase( Y320A): SEQ ID NO 78 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAKEFYISIETVGNNIVER YIDENGKERTREVEYLPTMFRHCKEESKYKDIYGKNCAPQKFPSMKDARDWMKRMED IGLEALGNINDFKLAYISDTYGSEIVYDRKFVRVANCDIEVTGDKFPDPMKAEYEIDAITH YDSIDDRFYVFDLLNSMYGSVSKWDAKLAAKLDCEGGDEVPQEILDRVIYMPFDNERD MLMEYINLWEQKRPAIFTGWNIEGFDVPYIMNRVKMILGERSMKRFSPIGRVKSKLIQN MYGSKEIYSIDGVSILDYLDLYKKFAFTNLPSFSLESVAQHETKKGKLPYDGPINKLRET NHQRYISYNIIDVESVQAIDKIRGFIDLVLSMSYYAKMPFSGVMSPIKTWDAIIFNSLKGE HKVIPQQGSHVKQSFPGAFVFEPKPIARRYIMSFDLTSLYPSIIRQVNISPETIRGQFKVHPI HEYIAGTAPKPSDEYSCSPNGWMYDKHQEGIIPKEIAKVFFQRKDWKKKMFAEEMNAE AIKKIIMKGAGSCSTKPEVERYVKFSDDFLNELSNYTESVLNSLIEECEKAATLANTNQL NRKILINSLYGALGNIHFRYYDLRNATAITIFGQVGIQWIARKINEYLNKVCGTNDEDFIA AGDTDSVYVCVDKVIEKVGLDRFKEQNDLVEFMNQFGKKKMEPMIDVAYRELCDYM NNREHLMHMDREAISCPPLGSKGVGGFWKAKKRYALNVYDMEDKRFAEPHLKIMGM ETQQSSTPKVVQEALEESIRRILQEGEESVQEYYKNFEKEYRQLDYKVIAEVKTANDIAK YDDKGWPGFKCPFHIRGVLTYRRAVSGLGVAPILDGNKVMVLPLREGNPFGDKCIAWP SGTELPKEIRSDVLSWIDHSTLFQKSFVKPLAGMCESAGMDYEEKASLDFLFG 3xFlag-NLS-T4 DNA polymerase( A737V): SEQ ID NO 79 MAPKRGKKGAVAEDGDELRTEPEAKKSKTAAKKNDKEAAGEGPALYEDPPDQKTSPS GKPATLKICSWNVDGLRAWIKKKGLDWVKEEAPDILCLQETKCSENKLPAELQELPGLS HQYWSAPSDKEGYSGVGLLSRQCPLKVSYGIGDEEHDQEGRVIVAEFDSFVLVTAYVP NAGRGLVRLEYRQRWDEAFRKFLKGLASRKPLVLCGDLNVAHEEIDLRNPKGNKKNA GFTPQERQGFGELLQAVPLADSFRHLYPNTPYAYTFWTYMMNARSKNVGWRLDYFLL SHSLLPALCDSKIRSKALGSDHCPITLYLAL APEX1: SEQ ID NO 80 MVRGSGKPIPNPLLGLDSTGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAF HSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAG VVAVEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALS GGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPL VDKYAADEDAFFADYAEAHQKLSELGFADAEFSRADPKKKRKVDPKKKRKVDPKKKR KV VStag-APEX2-NLS-NLS (Addgene#12 4617): SEQ ID NO 81 MERKISRIHLVSEPSITHFLQVSWEKTLESGFVITLTDGHSAWTGTVSESEISQEADDMA MEKGKYVGELRKALLSGAGPADVYTFNFSKESCYFFFEKNLKDVSFRLGSFNLEKVENP AEVIRELICYCLDTIAENQAKNEHLQKENERLLRDWNDVQGRFEKCVSAKEALETDLYK RFILVLNEKKTKIRSLHNKLLNAAQEREKDIKQEGETAICSEMTADRDPVYDESTDEESE NQTDLSGLASAAVSKDDSIISSLDVTDIAPSRKRRQRMQRNLGTEPKMAPQENQLQEKE KPDSSLPETSKKEHISAENMSLETLRNSSPEDLFDEI XRCC4: SEQ ID NO 82 MDAQTRRRERRAEKQAQWKAANGGSPPHMAYPYDVPDYAPPSRAQASNSAVDGTAG MGVPKFYRWISERYPCLSEVVKEHQIPEFDNLYLDMNGIIHQCSHPNDDDVHFRISDDKI FTDIFHYLEVLFRIIKPRKVFFMAVDGVAPRAKMNQQRGRRFRSAKEAEDKIKKAIEKG ETLPTEARFDSNCITPGTEFMARLHEHLKYFVNMKISTDKSWQGVTIYFSGHETPGEGEH KIMEFIRSEKAKPDHDPNTRHCLYGLDADLIMLGLTSHEAHFSLLREEVRFGGKKTQRV CAPEETTFHLLHLSLMREYIDYEFSVLKEKITFKYDIERIIDDWILMGFLVGNDFIPHLPHL HINHDALPLLYGTYVTILPELGGYINESGHLNLPRFEKYLVKLSDFDREHFSEVFVDLKW FESKVGNKYLNEAAGVAAEEARNYKEKKKLKGQENSLCWTALDKNEGEMITSKDNLE DETEDDDLFETEFRQYKRTYYMTKMGVDVVSDDFLADQAACYVQAIQWILHYYYHGV QSWSWYYPYHYAPFLSDIHNISTLKIHFELGKPFKPFEQLLAVLPAASKNLLPACYQHLM TNEDSPITEYYPPDFKTDLNGKQQEWEAWLIPFIDEKRLLEAMETCNHSLKKEERKRNQ HSECLMCWYDRDTEFIYPSPWPEKFPAIERCCTRYKIISLDAWRVDINKNKITRIDQKAL YFCGFPTLKHIRHKFFLKKSGVQVFQQSSRGENMMLEILVDAESDELTVENVASSVLGK SVFVNWPHLEEARWAVSDGETKFYLEEPPGTQKLYSGRTAPPSKWHLGDKEQSNWA KEVQGISEHYLRRKGIIINETSAVVYAQLLTGRKYQINQNGEVRLEKQWSKQWPFVYQ TIVKDIRAFDSRFSNIKTLDDLFPLRSMVFMLGTPYYGCTGEVQDSGDVITEGRIRVIFSIP CEPNLDALIQNQHKYSIKYNPGYVLASRLGVSGYLVSRFTGSIFIGRGSRRNPHGDHKAN VGLNLKFNKKNEEVPGYTKKVGSEWMYSSAAEQLLAEYLERAPELFSYIAKNSQEDVF YEDDIWPGENENGAEKVQEIITWLKGHPVSTLSRSSCDLQILDAAIVFKTFFFVEKCKQR KNNKKVRVTVKPHLLYRPLEQQHGVIPDRDAEFCLFDRVVNVRENFSVPVGLRGTIIGI KGANREADVLFEVLFDEEFPGGLTIRCSPGRGYRLPTSALVNLSHGSRSETGNQKLTAIV KPQPAVHQHSSSSSVSSGHLGALNHSPQSLFVPTQVPTKDDDEFCNIWQSLQGSGKMQY FQPTIQEKGAVLPQEISQVNQHHKSGFNDNSVKYQQRKHDPHRKFKEECKSPKAECWS QKMSNKQPNSGIENFLASLNISKENEVQSSHHGEPPSEEHLSPQSFAMGTRMLKEILKID GSNTVDHKNEIKQIANEIPVSSNRRDEYGLPSQPKQNKKLASYMNKPHSANEYHNVQS MDNMCWPAPSQIPPVSTPVTELSRICSLVGMPQPDFSFLRMPQTMTVCQVKLSNGLLVH GPQCHSENEAKEKAALFALQQLGSLGMNFPLPSQVFANYPSAVPPGTIPPAFPPPTGWD HYGSNYALGAANIMPSSSHLFGSMPWGPSVPVPGKPFHHTLYSGTMPMAGGIPGGVHN QFIPLQVTKKRVANKKNFENKEAQSSQATPVQTSQPDSSNIVKVSPRESSSASLKSSPIAQ PASSFQVETASQGHSISHHKSTPISSSRRKSRKLAVNFGVSKPSE V5tag-XRN1(Addge ne #66596): SEQ ID NO 83 MEQLNELELLMEKSFWEEAELPAELFQKKVVASFPRTVLSTGMDNRYLVLAVNTVQNK EGNCEKRLVITASQSLENKELCILRNDWCSVPVEPGDIIHLEGDCTSDTWIIDKDFGYLIL YPDMLISGTSIASSIRCMRRAVLSETFRSSDPATRQMLIGTVLHEVFQKAINNSFAPEKLQ ELAFQTIQEIRHLKEMYRLNLSQDEIKQEVEDYLPSFCKWAGDFMHKNTSTDFPQMQLS LPSDNSKDNSTCNIEVVKPMDIEESIWSPRFGLKGKIDVTVGVKIHRGYKTKYKIMPLEL KTGKESNSIEHRSQVVLYTLLSQERRADPEAGLLLYLKTGQMYPVPANHLDKRELLKLR NQMAFSLFHRISKSATRQKTQLASLPQIIEEEKTCKYCSQIGNCALYSRAVEQQMDCSSV PIVMLPKIEEETQHLKQTHLEYFSLWCLMLTLESQSKDNKKNHQNIWLMPASEMEKSGS CIGNLIRMEHVKIVCDGQYLHNFQCKHGAIPVTNLMAGDRVIVSGEERSLFALSRGYVK EINMTTVTCLLDRNLSVLPESTLFRLDQEEKNCDIDTPLGNLSKLMENTFVSKKLRDLIID FREPQFISYLSSVLPHDAKDTVACILKGLNKPQRQAMKKVLLSKDYTLIVGMPGTGKTT TICTLVRILYACGFSVLLTSYTHSAVDNILLKLAKFKIGFLRLGQIQKVHPAIQQFTEQEIC RSKSIKSLALLEELYNSQLIVATTCMGINHPIFSRKIFDFCIVDEASQISQPICLGPLFFSRRF VLVGDHQQLPPLVLNREARALGMSESLFKRLEQNKSAVVQLTVQYRMNSKIMSLSNKL TYEGKLECGSDKVANAVINLRHFKDVKLELEFYADYSDNPWLMGVFEPNNPVCFLNTD KVPAPEQVEKGGVSNVTEAKLIVFLTSIFVKAGCSPSDIGIIAPYRQQLKIINDLLARSIGM VEVNTVDKYQGRDKSIVLVSFVRSNKDGTVGELLKDWRRLNVAITRAKHKLILLGCVP SLNCYPPLEKLLNHLNSEKLIIDLPSREHESLCHILGDFQRE DNA2: SEQ ID NO 84 MEQKLISEEDLLRKRGILNLLRRSGKRRRSESGSDSFSGSGGDSSASPQFLSGSVLSPPPG LGRCLKAAAAGECKPTVPDYEIDKLLLANWGLPKAVLEKYHSFGVKKMFEWQAECLL LGQVLEGKNLVYSAPTSAGKTLVAELLILKRVLEMRKKALFILPFVSVAKEKKYYLQSL FQEVGIKVDGYMGSTSPSRHFSSLDIAVCTIERANGLINRLIEENKMDLLGMVVVDELH MLGDSHRGYLLELLLTKICYITRKSASCQADLASSLSNAVQIVGMSATLPNLELVASWL NAELYHTDFRPVPLLESVKVGNSIYDSSMKLVREFEPMLQVKGDEDHVVSLCYETICDN HSVLLFCPSKKWCEKLADIIAREFYNLHHQAEGLVKPSECPPVILEQKELLEVMDQLRRL Myc-POLQ-Flag(Addgen e# 73132): PSGLDSVLQKTVPWGVAFHHAGLTFEERDIIEGAFRQGLIRVLAATSTLSSGVNLPARRV IIRTPIFGGRPLDILTYKQMVGRAGRKGVDTVGESILICKNSEKSKGIALLQGSLKPVRSC LQRREGEEVTGSMIRAILEIIVGGVASTSQDMHTYAACTFLAASMKEGKQGIQRNQESV QLGAIEACVMWLLENEFIQSTEASDGTEGKVYHPTHLGSATLSSSLSPADTLDIFADLQR AMKGFVLENDLHILYLVTPMFEDWTTIDWYRFFCLWEKLPTSMKRVAELVGVEEGFLA RCVKGKVVARTERQHRQMAIHKRFFTSLVLLDLISEVPLREINQKYGCNRGQIQSLQQS AAVYAGMITVFSNRLGWHNMELLLSQFQKRLTFGIQRELCDLVRVSLLNAQRARVLYA SGFHTVADLARANIVEVEVILKNAVPFKSARKAVDEEEEAVEERRNMRTIWVTGRKGL TEREAAALIVEEARMILQQDLVEMGVQWNPCALLHSSTCSLTHSESEVKEHTFISQTKSS YKKLTSKNKSNTIFSDSYIKHSPNIVQDLNKSREHTSSFNCNFQNGNQEHQRCSIFRARK RASLDINKEKPGASQNEGKTSDKKVVQTFSQKTKKAPLNFNSEKMSRSFRSWKRRKHL KRSRDSSPLKDSGACRIHLQGQTLSNPSLCEDPFTLDEKKTEFRNSGPFAKNVSLSGKEK DNKTSFPLQIKQNCSWNITLTNDNFVEHIVTGSQSKNVTCQATSVVSEKGRGVAVEAEK INEVLIQNGSKNQNVYMKHHDIHPINQYLRKQSHEQTSTITKQKNIIERQMPCEAVSSYIN RDSNVTINCERIKLNTEENKPSHFQALGDDISRTVIPSEVLPSAGAFSKSEGQHENFLNISR LQEKTGTYTTNKTKNNHVSDLGLVLCDFEDSFYLDTQSEKIIQQMATENAKLGAKDTN LAAGIMQKSLVQQNSMNSFQKECHIPFPAEQHPLGATKIDHLDLKTVGTMKQSSDSHG VDILTPESPIFHSPILLEENGLFLKKNEVSVTDSQLNSFLQGYQTQETVKPVILLIPQKRTPT GVEGECLPVPETSLNMSDSLLFDSFSDDYLVKEQLPDMQMKEPLPSEVTSNHFSDSLCL QEDLIKKSNVNENQDTHQQLTCSNDESIIFSEMDSVQMVEALDNVDIFPVQEKNHTVVS PRALELSDPVLDEHHQGDQDGGDQDERAEKSKLTGTRQNHSFIWSGASFDLSPGLQRIL DKVSSPLENEKLKSMTINFSSLNRKNTELNEEQEVISNLETKQVQGISFSSNNEVKSKIEM LENNANHDETSSLLPRKESNIVDDNGLIPPTPIPTSASKLTFPGILETPVNPWKTNNVLQP GESYLFGSPSDIKNHDLSPGSRNGFKDNSPISDTSFSLQLSQDGLQLTPASSSSESLSIIDVA SDQNLFQTFIKEWRCKKRFSISLACEKIRSLTSSKTATIGSRFKQASSPQEIPIRDDGFPIKG CDDTLVVGLAVCWGGRDAYYFSLQKEQKHSEISASLVPPSLDPSLTLKDRMWYLQSCL RKESDKECSVVIYDFIQSYKILLLSCGISLEQSYEDPKVACWLLDPDSQEPTLHSIVTSFLP HELPLLEGMETSQGIQSLGLNAGSEHSGRYRASVESILIFNSMNQLNSLLQKENLQDVFR KVEMPSQYCLALLELNGIGFSTAECESQKHIMQAKLDAIETQAYQLAGHSFSFTSSDDIA EVLFLELKLPPNREMKNQGSKKTLGSTRRGIDNGRKLRLGRQFSTSKDVLNKLKALHPL PGLILEWRRITNAITKVVFPLQREKCLNPFLGMERIYPVSQSHTATGRITFTEPNIQNVPR DFEIKMPTLVGESPPSQAVGKGLLPMGRGKYKKGFSVNPRCQAQMEERAADRGMPFSI SMRHAFVPFPGGSILAADYSQLELRILAHLSHDRRLIQVLNTGADVFRSIAAEWKMIEPE SVGDDLRQQAKQICYGIIYGMGAKSLGEQMGIKENDAACYIDSFKSRYTGINQFMTETV KNCKRDGFVQTILGRRRYLPGIKDNNPYRKAHAERQAINTIVQGSAADIVKIATVNIQKQ LETFHSTFKSHGHREGMLQSDRTGLSRKRKLQGMFCPIRGGFFILQLHDELLYEVAEED VVQVAQIVKNEMESAVKLSVKLKVKVKIGASWGELKDFDVPGMDYKDDDDK SEQ ID NO 85 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAASKRKAPQETLNGGIT DMLTELANFEKNVSQAIHKYNAYRKAASVIAKYPHKIKSGAEAKKLPGVGTKIAEKIDE FLATGKLRKLEKIRQDDTSSSINFLTRVSGIGPSAARKFVDEGIKTLEDLRKNEDKLNHH QRIGLKYFGDFEKRIPREEMLQMQDIVLNEVKKVDSEYIATVCGSFRRGAESSGDMDVL LTHPSFTSESTKQPKLLHQVVEQLQKVHFITDTLSKGETKFMGVCQLPSKNDEKEYPHR RIDIRLIPKDQYYCGVLYFTGSDIFNKNMRAHALEKGFTINEYTIRPLGVTGVAGEPLPVD SEKDIFDYIQWKYREPKDRSE POLB: SEQ ID NO 86 MATGQDRVVALVDMDCFFVQVEQRQNPHLRNKPCAVVQYKSWKGGGIIAVSYEARAF GVTRSMWADDAKKLCPDLLLAQVRESRGKANLTKYREASVEVMEIMSRFAVIERASID EAYVDLTSAVQERLQKLQGQPISADLLPSTYIEGLPQGPTTAEETVQKEGMRKQGLFQW LDSLQIDNLTSPDLQLTVGAVIVEEMRAAIERETGFQCSAGISHNKVLAKLACGLNKPNR QTLVSHGSVPQLFSQMPIRKIRSLGGKLGASVIEILGIEYMGELTQFTESQLQSHFGEKNG SWLYAMCRGIEHDPVKPRQLPKTIGCSKNFPGKTALATREQVQWWLLQLAQELEERLT KDRNDNDRVATQLVVSIRVQGDKRLSSLRRCCALTRYDAHKMSHDAFTVIKNCNTSGI QTEWSPPLTMLFLCATKFSASAPSSSTDITSFLSSDPSSLPKVPVTSSEAKTQGSGPAVTA TKKATTSLESFFQKAAERQKVKEASLSSLTAPTQAPMSNSPSKPSLPFQTSQSTGTEPFFK QKSLLLKQKQLNNSSVSSPQQNPWSNCKALPNSLPTEYPGCVPVCEGVSKLEESSKATP AEMDLAHNSQSMHASSASKSVLEVTQKATPNPSLLAAEDQVPCEKCGSLVPVWDMPE POLH: HMDYHFALELQKSFLQPHSSNPQWSAVSHQGKRNPKSPLACTNKRPRPEGMQTLESFF KPLTH SEQ ID NO 87 MASRLLWRKVAGATVGPGPVPAPGRWVSSSVPASDPSDGQRRRQQQQQQQQQQQQQ PQQPQVLSSEGGQLRHNPLDIQMLSRGLHEQIFGQGGEMPGEAAVRRSVEHLQKHGLW GQPAVPLPDVELRLPPLYGDNLDQHFRLLAQKQSLPYLEAANLLLQAQLPPKPPAWAW AEGWTRYGPEGEAVPVAIPEERALVFDVEVCLAEGTCPTLAVAISPSAWYSWCSQRLVE ERYSWTSQLSPADLIPLEVPTGASSPTQRDWQEQLVVGHNVSFDRAHIREQYLIQGSRM RFLDTMSMHMAISGLSSFQRSLWIAAKQGKHKVQPPTKQGQKSQRKARRGPAISSWDW LDISSVNSLAEVHRLYVGGPPLEKEPRELFVKGTMKDIRENFQDLMQYCAQDVWATHE VFQQQLPLFLERCPHPVTLAGMLEMGVSYLPVNQNWERYLAEAQGTYEELQREMKKS LMDLANDACQLLSGERYKEDPWLWDLEWDLQEFKQKKAKKVKKEPATASKLPIEGAG APGDPMDQEDLGPCSEEEEFQQDVMARACLQKLKGTTELLPKRPQHLPGHPGWYRKL CPRLDDPAWTPGPSLLSLQMRVTPKLMALTWDGFPLHYSERHGWGYLVPGRRDNLAK LPTGTTLESAGWCPYRAIESLYRKHCLEQGKQQLMPQEAGLAEEFLLTDNSAIWQTVE ELDYLEVEAEAKMENLRAAVPGQPLALTARGGPKDTQPSYHHGNGPYNDVDIPGCWFF KLPHKDGNSCNVGSPFAKDFLPKMEDGTLQAGPGGASGPRALEINKMISFWRNAHKRIS SQMVVWLPRSALPRAVIRHPDYDEEGLYGAILPQVVTAGTITRRAVEPTWLTASNARPD RVGSELKAMVQAPPGYTLVGADVDSQELWIAAVLGDAHFAGMHGCTAFGWMTLQGR KSRGTDLHSKTATTVGISREHAKIFNYGRIYGAGQPFAERLLMQFNHRLTQQEAAEKAQ QMYAATKGLRWYRLSDEGEWLVRELNLPVDRTEGGWISLQDLRKVQRETARKSQWK KWEVVAERAWKGGTESEMFNKLESIATSDIPRTPVLGCCISRALEPSAVQEEFMTSRVN WVVQSSAVDYLHLMLVAMKWLFEEFAIDGRFCISIHDEVRYLVREEDRYRAALALQIT NLLTRCMFAYKLGLNDLPQSVAFFSAVDIDRCLRKEVTMDCKTPSNPTGMERRYGIPQG EALDIYQIIELTKGSLEKRSQPGP POLG: SEQ ID NO 88 MENYEALVGFDLCNTPLSSVAQKIMSAMHSGDLVDSKTWGKSTETMEVINKSSVKYSV QLEDRKTQSPEKKDLKSLRSQTSRGSAKLSPQSFSVRLTDQLSADQKQKSISSLTLSSCLI PQYNQEASVLQKKGHKRKHFLMENINNENKGSINLKRKHITYNNLSEKTSKQMALEED TDDAEGYLNSGNSGALKKHFCDIRHLDDWAKSQLIEMLKQAAALVITVMYTDGSTQLG ADQTPVSSVRGIVVLVKRQAEGGHGCPDAPACGPVLEGFVSDDPCIYIQIEHSAIWDQEQ EAHQQFARNVLFQTMKCKCPVICFNAKDFVRIVLQFFGNDGSWKHVADFIGLDPRIAA WLIDPSDATPSFEDLVEKYCEKSITVKVNSTYGNSSRNIVNQNVRENLKTLYRLTMDLC SKLKDYGLWQLFRTLELPLIPILAVMESHAIQVNKEEMEKTSALLGART KFT FQEAHFV AGERFLITSNNQLREILFGKLKLHLLSQRNSLPRTGLQKYPSTSEAVLNALRDLHPLPKIIL EYRQVHKIKSTFVDGLLACMKKGSISSTWNQTGTVTGRLSAKHPNIQGISKHPIQITTPK NFKGKEDKILTISPRAMFVSSKGHTFLAADFSQIELRILTHLSGDPELLKLFQESERDDVF STLTSQWKDVPVEQVTHADREQTKKVVYAVVYGAGKERLAACLGVPIQEAAQFLESFL QKYKKIKDFARAAIAQCHQTGCVVSIMGRRRPLPRIHAHDQQLRAQAERQAVNFVVQG SAADLCKLAMIHVFTAVAASHTLTARLVAQIHDELLFEVEDPQIPECAALVRRTMESLE QVQALELQLQVPLKVSLSAGRSWGHLVPLQEAWGPPPGPCRTESPSNSLAAPGSPASTQ PPPLHFSPSFCL POLN: SEQ ID NO 89 MASPCPEEAAMRREVVKRIETVVKDLWPTADVQIFGSFSTGLYLPTSDIDLVVFGKWER PPLQLLEQALRKHNVAEPCSIKVLDKATVPIIKLTDQETEVKVDISFNMETGVRAAEFIK NYMKKYSLLPYLILVLKQFLLQRDLNEVFTGGISSYSLILMAISFLQLHPRIDARRADENL GMLLVEFFELYGRNFNYLKTGIRIKEGGAYIAKEEIMKAMTSGYRPSMLCIEDPLLPGND VGRSSYGAMQVKQVFDYAYIVLSHAVSPLARSYPNRDAESTLGRIIKVTQEVIDYRRWI KEKWGSKAHPSPGMDSRIKIKERIATCNGEQTQNREPESPYGQRLTLSLSSPQLLSSGSSA SSVSSLSGSDVDSDTPPCTTPSVYQFSLQAPAPLMAGLPTALPMPSGKPQPTTSRTLIMTT NNQTRFTIPPPTLGVAPVPCRQAGVEGTASLKAVHHMSSPAIPSASPNPLSSPHLYHKHN GMKLSMKGSHGHTQGGGYSSVGSGGVRPPVGNRGHHQYNRTGWRRKKHTHTRDSLP VSLSR TENT4A: SEQ ID NO 90 MAASQTSQTVASHVPFADLCSTLERIQKSKGRAEKIRHFREFLDSWRKFHDALHKNHK DVTDSFYPAMRLILPQLERERMAYGIKETMLAKLYIELLNLPRDGKDALKLLNYRTPTG THGDAGDFAMIAYFVLKPRCLQKGSLTIQQVNDLLDSIASNNSAKRKDLIKKSLLQLITQ SSALEQKWLIRMIIKDLKLGVSQQTIFSVFHNDAAELHNVTTDLEKVCRQLHDPSVGLSD ISITLFSAFKPMLAAIADIEHIEKDMKHQSFYIETKLDGERMQMHKDGDVYKYFSRNGY NYTDQFGASPTEGSLTPFIHNAFKADIQICILDGEMMAYNPNTQTFMQKGTKFDIKRMV DNA Ligase 4: EDSDLQTCYCVFDVLMVNNKKLGHETLRKRYEILSSIFTPIPGRIEIVQKTQAHTKNEVID ALNEAIDKREEGIMVKQPLSIYKPDKRGEGWLKIKPEYVSGLMDELDILIVGGYWGKGS RGGMMSHFLCAVAEKPPPGEKPSVFHTLSRVGSGCTMKELYDLGLKLAKYWKPFHRK APPSSILCGTEKPEVYIEPCNSVIVQIKAAEIVPSDMYKTGCTLRFPRIEKIRDDKEWHEC MTLDDLEQLRGKASGKLASKHLYIGGDDEPQEKKRKAAPKMKKVIGIIEHLKAPNLTN VNKISNIFEDVEFCVMSGTDSQPKPDLENRIAEFGGYIVQNPGPDTYCVIAGSENIRVKNII LSNKHDVVKPAWLLECFKTKSFVPWQPRFMIHMCPSTKEHFAREYDCYGDSYFIDTDL NQLKEVFSGIKNSNEQTPEEMASLIADLEYRYSWDCSPLSMFRRHTVYLDSYAVINDLS TKNEGTRLAIKALELRFHGAKVVSCLAEGVSHVIIGEDHSRVADFKAFRRTFKRKFKILK ESWVTDSIDKCELQEENQYLI SEQ ID NO 91 MGSAACPRGALPELAPCCQPREQSQPHTRWDAGCGIQHPGGEEFRTLGGARAYRVPNS QEGRSSPTRFFPAPEGPAHCFVSSPDRAFWVSEEVQRLLLSNACQPKECNGVKIPVDASK PNPNDVEFDNLYLDMNGIIHPCTHPEDKPAPKNEDEMMVAIFEYIDRLFSIVRPRRLLYM AIDGVAPRAKMNQQRSRRFRASKEGMEAAVEKQRVREEILAKGGFLPPEEIKERFDSNC ITPGTEFMDNLAKCLRYYIADRLNNDPGWKNLTVILSDASAPGEGEHKIMDYIRRQRAQ PNHDPNTHHCLCGADADLIMLGLATHEPNFTIIREEFKPNKPKPCGLCNQFGHEVKDCE GLPREKKGKHDELADSLPCAEGEFIFLRLNVLREYLERELTMASLPFTFDVERSIDDWVF MCFFVGNDFLPHLPSLEIRENAIDRLVNIYKNVVHKTGGYLTESGYVNLQRVQMIMLAV GEVED SIFKKRKDDED SFRRRQKEKRKRMKRDQPAFTPSGILTPHALGSRNSPGSQ VAS NPRQAAYEMRMQNNSSPSISPNTSFTSDGSPSPLGGIKRKAEDSDSEPEPEDNVRLWEAG WKQRYYKNKFDVDAADEKFRRKVVQSYVEGLCWVLRYYYQGCASWKWYYPFHYAP FASDFEGIADMPSDFEKGTKPFKPLEQLMGVFPAASGNFLPPSWRKLMSDPDSSIIDFYPE DFAIDLNGKKYAWQGVALLPFVDERRLRAALEEVYPDLTPEETRRNSLGGDVLFVGKH HPLHDFILELYQTGSTEPVEVPPELCHGIQGKFSLDEEAILPDQIVCSPVPMLRDLTQNTV VSINFKDPQFAEDYIFKAVMLPGARKPAAVLKPSDWEKSSNGRQWKPQLGFNRDRRPV HLDQAAFRTLGHVMPRGSGTGIYSNAAPPPVTYQGNLYRPLLRGQAQIPKLMSNMRPQ DSWRGPPPLFQQQRFDRGVGAEPLLPWNRMLQTQNAAFQPNQYQMLAGPGGYPPRRD DRGGRQGYPREGRKYPLPPPSGRYNWN XRN: SEQ ID NO 92 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAASLKGKFFAFLPNPNTSS NKFFKSILEKKGATIVSSIQNCLQSSRKEVIILIEDSFVDSDMHLTQKDIFQREAGLNDVDE FLGKIEQSGIQCVKTSCITKWVQNDKFAFQKDDLIKFQPSIIVISDNADDGQSSTDKESEIS TDVESERNDDSNNKDMIQASKPLKRLLQEDKGRASLVTDKTKYKNNELIIGALKRLTKK YEIEGEKFRARSYRLAKQSMENCDFNVRSGEEAHTKLRNIGPSIAKKIQVILDTGVLPGL NDSVGLEDKLKYFKNCYGIGSEIAKRWNLLNFESFCVAAKKDPEEFVSDWTILFGWSYY DDWLCKMSRNECFAHLKKVQKALRGIDPECQVELQGSYNRGYSKCGDIDLLFFKPFCN DTTELAKIMETLCIKLYKDGYIHCFLQLTPNLEKLFLKRIVERFRTAKIVGYGERKRWYS SEIIKKFFMGVKLSPRELEELKEMKNDEGTLLIEEEEEETKLKPIDQYMSLNAKDGNYCR RLDFFCCKWDELGAGRIHYTGSKEYNRWIRILAAQKGFKLTQHGLFRNNILLESFNERRI FELLNLKYAEPEHRNIEWEKKTG 3xFlag_NLS PolIV: SEQ ID NO 93 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAALPSQSPAIFTVSRLNQT VRLLLEHEMGQVWISGEISNFTQPASGHWYFTLKDDTAQVRCAMFRNSNRRVTFRPQH GQQVLVRANITLYEPRGDYQIIVESMQPAGEGLLQQKYEQLKAKLQAEGLFDQQYKKP LPSPAHCVGVITSKTGAALHDILHVLKRRDPSLPVIIYPAAVQGDDAPGQIVRAIELANQ RNECDVLIVGRGGGSLEDLWSFNDERVARAIFTSRIPVVSAVGHETDVTIADFVADLRAP TPSAAAEVVSRNQQELLRQVQSTRQRLEMAMDYYLANRTRRFTQIHHRLQQQHPQLRL ARQQTMLERLQKRMSFALENQLKRTGQQQQRLTQRLNQQNPQPKIHRAQTRIQQLEYR LAETLRAQLSATRERFGNAVTHLEAVSPLSTLARGYSVTTATDGNVLKKVKQVKAGEM LTTRLEDGWIESEVKNIQPVKKSRKKVH 3xFlag _NLS _XseA: SEQ ID NO 94 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAPKKNEAPASFEKALSE LEQIVTRLESGDLPLEEALNEFERGVQLARQGQAKLQQAEQRVQILLSDNEDASLTPFTP DNE 3xFlag _NLS XseB: SEQ ID NO 95 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY 3xFlag-NLS- HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAI KKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSV LVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKK K SpCas9-NLS (Addgene#10 00000055) SEQ ID NO 96 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAI KKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRK VLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHK HYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAF KYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKK K 3xFlag-NLS-SpCas9(delta F916)-NLS: SEQ ID NO 97 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM 3xFlag-NLS-SpCas9(G915 F)-NLS: TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAI KKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAFFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA KSEQEIGKATAKYFFYSNININFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSV LVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKK K SEQ ID NO 98 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAI KKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIKRPLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA KSEQEIGKATAKYFFYSNININFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSV LVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKK K 3xFlag-NLS-SpCas9(Q920 P)-NLS: SEQ ID NO 99 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAI KKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD 3xFlag-NLS-SpCas9(F916 P)-NLS DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGPIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSV LVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKK K SEQ ID NO 100 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAI KKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIARQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSV LVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKK K 3xFlag-NLS-SpCas9(R918 A) -NLS SEQ ID NO 101 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAI KKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG GLSELDKAGFIKPQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSV LVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF 3xFlag-NLS-SpCas9(R919 P)-NLS SEQ ID NO SEQUENCE ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKK K SEQ ID NO 102 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD N R E K IE K IL TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED ILEDIVL TL TLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFACRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAI KKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQITQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS ELDKAMFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK DFQFYKVREINKYHHAHDA YLNA VVGT ALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKV LSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV A YSVL V VAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFK YFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQ 4KKKK 3 xFlag-NL S-SpCas9-NLS(N690C T769I G915M N980K): LZ3Cas9Add gene# 140561: SEQ ID NO 103 MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVEASMKRNYILGLDIGITSVGYGIID YETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISR NSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFI DTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY NALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVT STGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEE IEQISNLKGYTGTHNLSLKAINLILDEL WHTNDNQIAIFNRLKL VPKKVDLSQQKEIPTTL VDDFIL SP WKRSFIQ SIK VIN AIIKKYGLPNDIIIEL AREKNSKD AQKMINEMQKRNRQTN ERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVS FDNSFNNKVL VKQEENSKKGNRTPFQYL S S SD SKI SYETFKKHILNLAKGKGRI SKTKKE YLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSF LRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVIvIENQMFEEKQAES MPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYY EETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDV YLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKIN GELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILG NL YEVKSKKHPQIIKKGRSGGGEGRGSLL TCGDVEENPGPMVSKGEELFTGVVPIL VEL DGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDH MKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVL LPDNHYL STQ SAL SKDPNEKRDHMVLLEF VTAAGITL GMDELYK 3 xFlag-NL S-SaCas9-P2A-EGFP: SEQ ID NO 104 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQF FIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKN FnCasl2a-NLS- LFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEAT _(ΓIIKSFKGWTTYFKGF) HENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELT FDIDYKTSEVNORVFSLDEVFEIANFNNYLNOSGITKFNTIIGGKFVNGENTKRKGINEYI 3xHA(addge ne#64709): NLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKT VEEKSIKETL SLLFDDLKAQKLDL SKIYFKNDKSLTDL SQQ VFDD YS VIGT AVLEYITQQI APKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIP MIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHIS QSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLA NGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPG ANKMLPKVFFSAKSIKFYNPSEDILRIRNHS IH I KNGSPQKG YEKF EF NIED CRKFIDFYK QSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYL FQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHP AKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLK EKAND VHIL SIDRGERHLAYYTL VD GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDR DSARKDWKKINNIKEMKEGYLSQWHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQV YQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFT SKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGK WTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDK KFF AKL TSVLNTILQMRNSKTGTELDYLISPV ADVNGNFFDSRQAPKNMPQDADANGA YHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNKRPAATKKAGQAKKKK GSYPYDVPDY A YPYDVPDY A YPYDVPDY A SEQ ID NO 105 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTY ADQ CLQL VQLD WENL SAAID SYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAI NKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVF SAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFP FYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLF KQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHK KLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKE LSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESN EVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNK EKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIP KCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQK GYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAE KEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAEL FYRPKSRMKRMAHRL GEKMLNKKLKDQKTPIPDTLYQELYDYVNHRL SHDL SDEARA LLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIG IDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSWGTIKD LKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNC LVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFV WKTIKNHE SRKHFLEGFDFLHYD VKTGDFILHFKNINRNL SFQRGLPGFMI’AWDI VFEK NETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKL LENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMD ADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNKRPAATKKAGQ 4KKKK GSYPYD VPDYAYPYD VPDYAYPYD VPDYA AsCas 12a-NLS-3xHA(addge ne#69982): SEQ ID NO 106 MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYL SFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFK KDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTR YISNMDIFEKVDAIFDKHEVQEIKEKILNSDYD VEDFFEGEFFNFVLTQEGID VYNAIIGGF VTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVF RNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAE YDDIHLKKKA VVTEKYEDDRRKSFKKIGSFSLEQLQEY ADADLSVVEKLKEIIIQKVDEI YKVYGSSEKLFDADFVLEKSLKKNDAWAIMKDLLDSVKSFENYIKAFFGEGKETNRD ESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETD YRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFS KKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNF SETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGT PNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEEL VVHP ANSPIANKNPDNPKK TTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGER NLLYIVWDGKGNIVEOYSLNEIINNFNGIRIKTDYHSLLDKKEKERPE ARQNWTSIENIK HLbCas 12a-NLS-3xHA(addge ne#69988): ELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNY MVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKT KYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRN PKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLML QMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWA IGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQAKKKKGSYPYDVP DYAYPYD VPDYAYPYD VPDYA SEQ ID NO 107 GGGSGGGSGGGS 3xGS: SEQ ID NO 108 SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SGGS)2-XTEN-(SGGS)2 SEQ ID NO 109 AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (H4)2: SEQ ID NO 110 GSDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAA 3xFlag-NLS: SEQ ID NO 111 GSDYKDHDGDYKDHDIDYKDDDDKGIHGVPAA 3xFlag: SEQ ID NO 112 GSGSEAAAKEAAAKEAAAKEAAAKALEAAAAKEAAAKEAAAKEAAAKGSGSAAAKE AAAKEAAAKEAAAKGSGS (H4)3: SEQ ID NO AGSGGSGGSGGSPVPSTPPTNSSSTPPTPSPSPVPSTPPTNSSSTPPTPSPSPVPSTPPTNSSS TPPTPSPSAS 113 GPcPcPc: SEQ ID NO 114 AGSGGSGGSGGSPVPSTPPTPSPSTPPTPSPSGGSGNSSGSGGSPVPSTPPTPSPSTPPTPSPS AS GPGcP: SEQ ID NO 115 AGSGGSGGSGGSPVPSTPPTPSPSTPPTPSPSIQRTPKIQVYSRHPAENGKSNFLNCYVSGF HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQP KIVKWDRDGGSGGSGGSGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDL LKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRD PVPSTPPTPSPSTPPTPSPSAS GPbGbP: SEQ ID NO 116 AGSGGSGGSGGSPVPSTPPTPSPSTPPTPSPSDGRYSLTYIYTGLSKHVEDVPAFQALGSL NDLQFFRYNSKDRKSQPMGLWRQVEGMED WKQD SQLQKAREDIFMETLKDIVEYYND SNGSHVLQGRFGCEIENNRSSGAFWKYYYDGKDYIEFNKEIPAWVPFDPAAQITKQKW EAEPVYVQRAKAYLEEECPATLRKYLKYSKNILDRQDPPSWVTSHQAPGEKKKLKCL AYDFYPGKID VHWTRAGEVQEPELRGD VLHNGNGTYQSW V WAVPPQDTAPYS CHVQ HSSLAQPLVVPWEASPVPSTPPTPSPSTPPTPSAS GPZP: SEQ ID NO 117 AGSGGSGGSGGSGGSGGSGGSGGSDGRYSLTYIYTGLSKHVEDVPAFQALGSLNDLQFF RYNSKDRKSQPMGL WRQ VEGMED WKQD SQLQK AREDIFMETLKDIVEYYND SNGSH V LQGRFGCEIENNRSSGAFWKYYYDGKDYIEFNKEIPAWVPFDPAAQITKQKWEAEPVY VQRAKAYLEEECPATLRKYLKYSKNILDRQDPPSWVTSHQAPGFKKK1 KCT AYDFYP GGZGZP: GKIDVHWTRAGEVQEPELRGDVLHNGNGTYQSWVVVAVPPQDTAPYSCHVQHSSLAQ PLVVPWEASGGSGGSGGSGGSDGRYSLTYIYTGLSKHVEDVPAFQALGSLNDLQFFRYN SKDRKSQPMGLWRQVEGMEDWKQDSQLQKAREDIFMETLKDIVEYYNDSNGSHVLQG RFGCEIENNRSSGAFWKYYYDGKDYIEFNKEIPAWVPFDPAAQITKQKWEAEPVYVQR AKAYLEEECPATLRKYLKYSKNILDRQDPPSWVTSHQAPGEKKKLKCLAYDFYPGKID VHWTRAGEVQEPELRGDVLHNGNGTYQSWVWAVPPQDTAPYSCHVQHSSLAQPLW PWEASPVPSTPPTPSPSTPPTPSPSAS

The skilled person in the art would appreciate that the amino acid sequences, peptides, polypeptides, nucleases, polymerases, blunting enzymes, guide RNAs, and single guide RNAs disclosed herein can be encoded by nucleic acid molecules. The skilled person in the art would also appreciate that vectors comprising these nucleic acid molecules could be used as vehicles to carry the genetic materials into cells. The vector can be a plasmid and is generally made of a DNA sequence that consists of an insert and a larger sequence that serves as the “backbone” of the vector.

EXAMPLES

While several experimental Examples are contemplated, these Examples are intended nonlimiting.

Example 1 Indels Editing in PCSK9 Gene Using Cas9 and Blunting Enzymes

To test for the efficiency of inducing indels in a target gene using Cas9 and blunting enzymes, Cas9 and PCSK9 exon 12 targeting sgRNA were co-transferred into cultured mammalian cells in combination with DNA polymerase ₁₁ (POLM), EXOG, T4 DNA polymerase (T4pol), DNA polymerase λ (POLL), MGME1, RecJ exonuclease (RecJ) or Nuclease S1 (nucS). Cas9 and sgRNA alone served as the negative control. The occurrence of indels for the control and each of the combinations was measured. HEK293T cells were used. Results for each of the combinations are presented in FIGS. 11A-11H and comparisons between the control and blunting enzymes are presented in FIGS. 12A-12B.

POLM (FIG. 11B), T4pol (FIG. 1 ID) and POLL (FIG. 11E) were found to increase the percentage of +1 insertion from 14.4% to 19.6%, 14.4% to 36.75%, and 14.4% to 39.55%, respectively. EXOG (FIG. 11C), MGME1 (FIG. 11F) and RecJ (FIG. 11G) were found to increase the percentage of -1 deletion from 4.3% to 5.05%, 4.3% to 6.35%, and 4.3% to 5.5% respectively.

POLL (FIG. 12A) and T4pol (FIG. 12B) were found to increase the +1 insertion frequency from 9.4% to 35.0% and 9.4% to 30.3% respectively.

Example 2 Indels Editing in GYPB Gene Using Cas9 and Blunting Enzymes

To test for the efficiency of inducing indels in a target gene using Cas9 and blunting enzymes, Cas9 and GYPB targeting sgRNA were co-transferred into cultured mammalian cells in combination with DNA polymerase ₁₁ (POLM), EXOG, T4 DNA polymerase (T4pol), DNA polymerase λ (POLL), MGME1, RecJ exonuclease (RecJ) or Nuclease S 1 (nucS). Cas9 and sgRNA alone served as the negative control. The occurrence of indels for the control and each of the combinations was measured. Results for each of the combinations are presented in FIGS. 13A-13H.

POLM (FIG. 13B), T4pol (FIG. 13D) and POLL (FIG. 13E) were found to increase the percentage of+1 insertion mutations from 10.5% to 13.7%, 10.5% to 13.4%, and 10.5% to 22.1% respectively. T4 polymerase (FIG. 13D) was found to increase the percentage of -1 deletion mutations from 1.7% to 9.05%

Example 3 Indels Editing in TPH2 Gene Using Cas9 and Blunting Enzymes

To test for the efficiency of inducing indels in a target gene using Cas9 and blunting enzymes, Cas9 and TPH2 targeting sgRNA were co-transferred into cultured mammalian cells in combination with POLM, EXOG, T4 polymerase, DNA polymerase λ (POLL), MGME1, RecJ exonuclease (RecJ) or Nuclease S 1 (nucS). Cas9 and sgRNA alone served as the negative control. The occurrence of indels for the control and each of the combinations was measured. Results for each of the combinations are presented in FIGS. 14A-14H.

DNA polymerase µ (POLM) (FIG. 14B) and DNA polymerase I_(v) (POLL) (FIG. 14E) were found to increase the percentage of +1 insertion mutations from 0% to 12.5% and 0% to 2.95 respectively. T4 DNA polymerase (T4pol) (FIG. 14D) were found to increase the percentage of -1 deletion mutations from 13.2% to 36.7%.

One skilled in the art will appreciate further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. 

1-73. (canceled)
 74. A composition comprising: (a) a target specific nuclease, wherein a target comprises a double stranded DNA (dsDNA); (b) a double strand break (DSB)-end blunting enzyme; and (c) a single guide RNA (sgRNA) comprising a variable sequence of nucleic acids for target recognition and one or more stem loops, wherein the nucleic acid sequence of the stem loop is at least 90% identical to a nucleic acid transcribed from a lowercase sequence of SEQ ID NOs: 54-64 of Table
 1. 75. The composition of claim 74, wherein the nucleic acid sequence of the stem loop is at least 95% identical to a nucleic acid transcribed from a lowercase sequence of SEQ ID NOs: 54-64 of Table
 1. 76. The composition of claim 74, wherein the nucleic acid sequence of the sgRNA is about 5 to about 75 nucleotides in length.
 77. The composition of claim 74, wherein the sgRNA comprises a variable sequence of nucleic acid for target recognition of about 17 to about 23 nucleotides in length.
 78. The composition of claim 74, further comprising a MS2-binding protein linked to the sgRNA by the one or more MS2 stem loops of the sgRNA.
 79. The composition of claim 74, wherein the nuclease induces staggered ends upon cleaving the dsDNA, optionally wherein the nuclease is an altered scissile variant.
 80. The composition of claim 74, wherein the target specific nuclease is selected from the group consisting of Cas12a, LbCas12a, FnCas12a, AsCas12a, Cas9, SpCas9, SaCas9, LZ3Cas9 (SpCas9 N690C, T769I, G915M, and N980K mutant), SpCas9 G915F mutant, SpCas9 AF916 mutant, SpCas9 F916P mutant, SpCas9 R918A mutant, SpCas9 R919P mutant, SpCas9 Q920P mutant, Cascφ, and the double combinations of Cas9 nickase, zinc finger nuclease (ZFN), and TAL Effector Nuclease (TALEN).
 81. The composition of claim 74, wherein the nuclease comprises an amino acid sequence at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106.
 82. The composition of claim 74, wherein the nuclease specifically binds to a protospacer-adjacent motif (PAM), optionally wherein the PAM is selected from the group consisting of NNNNGATT, NNNNGNNN, NNG, NG, NGAN, NGNG, NGAG, NGCG, NAAG, NGN, NRN, NNGRRN, NNNRRT, TTTN, TTTV, TYCV, TATV, TYCV, TATV, TTN, KYTV, TYCV, TATV, and TBN.
 83. The composition of claim 74, wherein the DSB-end blunting enzyme is a polymerase.
 84. The composition of claim 83, wherein the polymerase is selected from the group consisting of DNA polymerase λ (lambda) (POLL), DNA polymerase µ(mu) (POLM), DNA polymerase β(beta) (POLB), DNA polymerase γ(gamma) (POLG), DNA polymerase

(iota) (POLI), DNA polymerase η (eta) (POLH), TENT4A, DNA polymerase v (nu) (POLN), DNA Ligase 4, DNTT, XRCC4, DNA Polymerase IV, fungi pol IV-like DNA polymerase, DNA polymerase/3′-5′ exonuclease Pol X, and T4 DNA polymerase (T4pol).
 85. The composition of claim 74, wherein the DSB-end blunting enzyme is a single-strand DNA specific nuclease.
 86. The composition of claim 85, wherein the single-strand DNA specific nuclease is selected from the group consisting of MGME1, FEN1, DNA2, XRN2, EXOG, EXO1, AP endonuclease, RecJ exonuclease (RecJ), XseA, XseB, nuclease S1 (nucS), P1 nuclease, Artemis, T4 DNA polymerase (T4pol), and Csm1, optionally wherein the DSB-end blunting enzyme is modified with a protein tag comprising Myc, Flag or VStag.
 87. The composition of claim 74, wherein the DSB-end blunting enzyme comprises an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-94.
 88. The composition of claim 74, wherein the DSB-end blunting enzyme is covalently bound to the nuclease by a linker.
 89. The composition of claim 88, wherein the linker is a peptide.
 90. The composition of claim 74, wherein the composition further comprises an inhibitor of the microhomology-mediated end joining (MMEJ) pathway.
 91. The composition of claim 90, wherein the MMEJ pathway inhibitor is a CtIP or MRN inhibitor, optionally wherein the CtIP inhibitor is selected from KLHL15 and PIN1, and optionally wherein the MRN inhibitor is selected from E1b55K and E40rf6.
 92. A composition comprising: (a) a first nucleic acid molecule encoding a target specific nuclease, wherein a target comprises a double stranded DNA (dsDNA); (b) a second nucleic acid molecule encoding a DSB-end blunting enzyme; and (c) a third nucleic acid molecule encoding a sgRNA comprising a variable sequence of nucleic acid for target recognition and one or more stem loops, wherein the nucleic acid sequence of the stem loop is at least 90% identical to a nucleic acid transcribed from a lowercase sequence of SEQ ID NOs: 54-64 of Table
 1. 93. A method of inserting or deleting one or more single base pairs in a double-stranded DNA (dsDNA), the method comprising: (a) cleaving the dsDNA at a target site with a target specific nuclease and a single guide RNA (sgRNA) comprising a variable sequence of nucleic acid for target recognition and one or more stem loops, wherein the nucleic acid sequence of the stem loop is at least 90% identical to a nucleic acid transcribed from a lowercase sequence of SEQ ID NOs: 54-64 of Table 1, and wherein the cleavage results in overhangs on both dsDNA ends; (b) inserting a nucleotide complementary to the overhanging nucleotide on both of the dsDNA ends using a double strand break (DSB)-end blunting enzyme, or removing the overhanging nucleotide on both of the dsDNA ends using the DSB-end blunting enzyme; and (c) ligating the dsDNA ends together, thereby inserting or deleting a single base pair in the dsDNA, optionally wherein: the nucleic acid sequence of the stem loop is at least 90% identical to a nucleic acid transcribed from a lowercase sequence of SEQ ID NOs: 54-64 of Table 1, the nucleic acid sequence of the sgRNA is more than about 5 to about 75 nucleotides in length, the nuclease is an altered scissile variant, the nuclease comprises an amino acid sequence at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-106, the amino acid sequence specifically binds to a protospacer-adjacent motif (PAM), the DSB-end blunting enzyme is a polymerase, the DSB-end blunting enzyme is a single-strand DNA specific nuclease, the DSB-end blunting enzyme comprises an amino acid sequence at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-94, the DSB-end blunting enzyme is covalently bound to the target specific nuclease by a linker, the dsDNA is in a cell, and/or the composition further comprises an inhibitor of the microhomology-mediated end joining (MMEJ) pathway.
 94. A method of treating a disease caused by a frameshift mutation in a dsDNA in a subject, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising: (a) a target specific nuclease, wherein a target comprises a double stranded DNA (dsDNA); (b) a double strand break (DSB)-end blunting enzyme; and (c) a single guide RNA (sgRNA) comprising a variable sequence of nucleic acid for target recognition and one or more stem loops, wherein the nucleic acid sequence of the stem loop is at least 90% identical to a nucleic acid transcribed from a lowercase sequence of SEQ ID NOs: 54-64 of Table 1, and wherein at least one base pair in the dsDNA is inserted or deleted within the frameshift mutation. 